Method of immunosuppression

ABSTRACT

This invention relates to tricyclo compounds useful for treatment and prevention of resistance by transplantation, graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases, infectious diseases, and the like, which can be represented by the following formula: ##STR1## to a process for their production, to a pharmaceutical composition containing the same and to a use thereof.

This application is a Continuation of application Ser. No. 08/753,950,filed on Dec. 3, 1996, now U.S. Pat. No. 5,830,717, which is aContinuation of Ser. No. 08/450,412, filed May 25, 1995, now U.S. Pat.No. 5,624,842, which is a Divisional of Ser. No. 07/824,384, filed Jan.23, 1992, abandoned, which is a Divisional of Ser. No. 07/491,205, filedMar. 9, 1990, now U.S. Pat. No. 5,110,811, which is a Continuation ofSer. No. 06/868,749, filed May 30, 1986, now U.S. Pat. No. 4,929,611,which is a Continuation in Part of Ser. No. 06/799,855, filed Nov. 20,1985, now U.S. Pat. No. 4,894,366.

This invention relates to novel tricyclo compounds havingpharmacological activities, to a process for their production and to apharmaceutical composition containing the same.

More particularly, it relates to novel tricyclo compounds, which havepharmacological activities such as immunosuppressive activity,antimicrobial activity, and the like, to a process for their production,to a pharmaceutical composition containing the same and to a usethereof.

Accordingly, one object of this invention is to provide a novel tricyclocompounds, which are useful for treatment and prevention of resistanceby transplantation, graft-versus-host diseases by medulla ossiumtransplantation, autoimmune diseases, infectious diseases, and the like.

Another object of this invention is to provide a process for productionof the tricyclo compounds by fermentation processes and syntheticprocesses.

A further object of this invention is to provide a pharmaceuticalcomposition containing, as active ingredients, the tricyclo compounds.

Still further object of this invention is to provide a use of thetricyclo compounds for manufacturing a medicament for treating andpreventing resistance by transplantation, graft-versus-host diseases bymedulla ossium transplantation, autoimmune diseases, infectiousdiseases, and the like.

With respect to the present invention, it is to be noted that thisinvention is originated from and based on the first and new discovery ofnew certain specific compounds, FR-900506, FR-900520, FR-900523 andFR-900525 substances. In more detail, the FR-900506, FR-900520,FR-900523 and FR-900525 substances were firstly and newly isolated inpure form from culture broths obtained by fermentation of new speciesbelonging to genus Streptomyces.

And, as a result of an extensive study for elucidation of chemicalstructures of the FR-900506, FR-900520, FR-900523 and FR-900525substances, the inventors of this invention have succeeded indetermining the chemical structures thereof and in producing thetricyclo compounds of this invention.

The new tricyclo compounds of this invention can be represented by thefollowing general formula: ##STR2## wherein R¹ is hydroxy or protectedhydroxy,

R² is hydrogen, hydroxy or protected hydroxy,

R³ is methyl, ethyl, propyl or allyl,

n is an integer of 1 or 2, and the symbol of a line and dotted line is asingle bond or a double bond,

and salts thereof.

Among the object compound (I), the following four specific compoundswere found to be produced by fermentation.

(1) The compound (I) wherein R¹ and R² are each hydroxy, R³ is allyl, nis an integer of 2, and the symbol of a line and dotted line is a singlebond, which is entitled to the FR-900506 substance;

(2) The compound (I) wherein R¹ and R² are each hydroxy, R³ is ethyl, nis an integer of 2, and the symbol of a line and dotted line is a singlebond, which is entitled to the FR-900520 substance (another name: the WS7238A substance);

(3) The compound (I) wherein R¹ and R² are each hydroxy, R³ is methyl, nis an integer of 2, and the symbol of a line and dotted line is a singlebond, which is entitled to the FR-900523 substance (another name: the WS7238B substance); and

(4) The compound (I) wherein R¹ and R² are each hydroxy, R³ is allyl, nis an integer of 1, and the symbol of a line and dotted line is a singlebond, which is entitled to the FR-900525 substance.

With respect to the tricyclo compounds (I) of this invention, it is tobe understood that there may be one or more conformer(s) orstereoisomeric pairs such as optical and geometrical isomers due toasymmetric carbon atom(s) and double bond(s), and such isomers are alsoincluded within a scope of this invention.

According to this invention, the object tricycle compounds (I) can beprepared by the following processes. ##STR3## in which R¹, R², R³, n andthe symbol of a line and dotted line are each as defined above,

R_(a) ¹ and R_(a) ² are each protected hydroxy,

R_(b) ¹ is protected carboxy(lower)alkylcarbamoyloxy,

R_(c) ¹ is carboxy(lower)alkylcarbamoyloxy, and

R_(b) ² is a leaving group.

Particulars of the above definitions and the preferred embodimentsthereof are explained in detail as follows.

The term "lower" used in the specification is intended to mean 1 to 6carbon atoms, unless otherwise indicated.

Suitable hydroxy-protective group in the "protected hydroxy" mayinclude:

1-(lower alkylthio)(lower, alkyl such as lower alkylthiomethyl (e.g.methylthiomethyl, ethylthiomethyl, propylthiomethyl,isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl,hexylthiomethyl, etc.), and the like, in which the preferred one may beC₁ -C₄ alkylthiomethyl and the most preferred one may bemethylthiomethyl;

trisubstituted silyl such as tri(lower)alkylsilyl (e.g. trimethylsilyl,triethylsilyl, tributylsilyl, tert-butyl-dimethylsilyl,tri-tert-butylsilyl, etc.), lower alkyl-diarylsilyl (e.g.methyl-diphenylsilyl, ethyl-diphenylsilyl, propyl-diphenylsilyl,tert-butyl-diphenylsilyl, etc.), and the like, in which the preferredone may be tri(C₁ -C₄)alkylsilyl and C₁ -C₄ alkyl-diphenylsilyl, and themost preferred one may be tert-butyl-dimethylsilyl andtert-butyl-diphenylsilyl;

acyl such as aliphatic acyl, aromatic acyl and aliphatic acylsubstituted with aromatic group, which are derived from carboxylic,sulfonic and carbamic acids; and the like.

The aliphatic acyl may include lower alkanoyl which may have one or moresuitable substituent(s) such as carboxy (e.g. formyl, acetyl, propionyl,butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl,carboxyacetyl, carboxypropionyl, carboxybutyryl, carboxyhexanoyl, etc.),cyclo(lower)alkyloxy(lower)alkanoyl which may have one or more suitablesubstituent(s) such as lower alkyl (e.g. cyclopropyloxyacetyl,cyclobutyloxypropionyl, cycloheptyloxybutyryl, menthyloxyacetyl,menthyloxypropionyl, menthyloxybutyryl, menthyloxyheptanoyl,menthyloxyhexanoyl, etc.), camphorsulfonyl, lower alkylcarbamoyl havingone or more suitable substituent(s) such as carboxy and a protectedcarboxy, for example, carboxy(lower)alkylcarbamoyl (e.g.carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl,carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl,etc.), protected carboxy(lower)alkylcarbamoyl such astri(lower)alkylsilyl(lower)alkoxycarbonyl(lower)alkylcarbamoyl (e.g.trimethylsilylmethoxycarbonylethylcarbamoyl,trimethylsilylethoxycarbonylpropylcarbamoyl,triethylsilylethoxycarbonylpropylcarbamoyl,tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl,trimethylsilylpropoxycarbonylbutylcarbamoyl, etc.), and the like.

The aromatic acyl may include aroyl which may have one or more suitablesubstituent(s) such as nitro (e.g. benzoyl, toluoyl, xyloyl, naphthoyl,nitrobenzoyl, dinitrobenzoyl, nitronaphthoyl, etc.), arenesulfonyl whichmay have one or more suitable substituent(s) such as halogen (e.g.benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl,fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl,iodobenzenesulfonyl, etc.), and the like.

The aliphatic acyl substituted with aromatic group may includear(lower)alkanoyl which may have one or more suitable substituent(s)such as lower alkoxy and trihalo(lower)alkyl (e.g. phenylacetyl,phenylpropionyl, phenylbutyryl,2-trifluoromethyl-2-methoxy-2-phenylacetyl,2-ethyl-2-trifluoromethyl-2-phenylacetyl,2-trifluoromethyl-2-propoxy-2-phenylacetyl, etc.), and the like.

The more preferred acyl group thus defined may be C₁ -C₄ alkanoyl whichmay have carboxy, cyclo(C₅ -C₆)-alkyloxy(C₁ -C₄)alkanoyl having two (C₁-C₄)alkyl groups on the cycloalkyl moiety, camphorsulfonyl, carboxy(C₁-C₄)-alkylcarbamoyl, tri(C₁ -C₄)alkylsilyl(C₁ -C₄)alkoxycarbonyl-(C₁-C₄)alkylcarbamoyl, benzoyl which may have one or two nitro,benzenesulfonyl having halogen, phenyl(C₁ -C₄)alkanoyl having C₁ -C₄alkoxy and trihalo(C₁ -C₄)alkyl, and the most preferred one may beacetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl,nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and2-trifluoromethyl-2-methoxy-2-phenylacetyl.

Suitable "protected carboxy(lower)alkylcarbamoyl" and"carboxy(lower)alkylcarbamoyl" moieties of the "protectedcarboxy(lower)alkylcarbamoyloxy" and "carboxy(lower)alkylcarbamoyloxy"groups may include the same as those exemplified in the explanation ofthe hydroxy-protective group mentioned above.

Suitable "leaving group" may include hydroxy, acyloxy in which the acylmoiety may be those as exemplified above, and the like.

The processes for production of the tricyclo compounds (I) of thisinvention are explained in detail in the following.

[I] Fermentation Processes

The FR-900506, FR-900520, FR-900523 and FR-900525 substances of thisinvention can be produced by fermentation of FR-900506, FR-900520,FR-900523 and/or FR-900525 substance(s)-producing strains belonging tothe genus Streptomyces such as Streptomyces tsukubaensis No. 9993 andStreptomyces hygroscopicus subsp. yakushimaensis No. 7238 in a nutrientmedium.

Particulars of microorganisms used for the production of the FR-900506,FR-900520, FR-900523 and FR-900525 substances are explained in thefollowing.

[A] The FR-900506, FR-900520 and FR-900525 substances of this inventioncan be produced by fermentation of a FR-900506, FR-900520 and/orFR-900525 substance(s)-producing strain belonging to the genusStreptomyces such as Streptomyces tsukubaensis No. 9993 in a nutrientmedium.

THE MICROORGANISM

The microorganism which can be used for the production of the FR-900506,FR-900520 and/or FR-900525 substances is FR-900506, FR-900520 and/orFR-900525 substance(s)-producing strain belonging to the genusStreptomyces, among which Streptomyces tsukubaensis No. 9993 has beennewly isolated from a soil sample collected at Toyosato-cho,Tsukuba-gun, Ibaraki Prefecture, Japan.

A lyophilized sample of the newly isolated Streptomyces tsukubaensis No.9993 has been deposited with the Fermentation Research Institute, Agencyof Industrial Science and Technology (No. 1-3, Higashi 1-chome,Yatabemachi Tsukuba-gun, Ibaraki Prefecture, Japan) under the depositnumber of FERM P-7886 (deposited date: October 5th, 1984), and thenconverted to Budapest Treaty route of the same depository on Oct. 19,1985 under the new deposit number of FERM BP-927.

It is to be understood that the production of the novel FR-900506,FR-900520 and/or FR-900525 substance(s) is not limited to the use of theparticular organism described herein, which is given for theillustrative purpose only. This invention also includes the use of anymutants which are capable of producing the FR-900506, FR-900520 and/orFR-900525 substances including natural mutants as well as artificialmutants which can be produced from the described organism byconventional means such as irradiation of X-rays, ultra-violetradiation, treatment with N-methyl-N'-nitro-N-nitrosoguanidine,2-aminopurine, and the like.

The Streptomyces tsukubaensis No. 9993 has the following morphological,cultural, biological and physiological characteristics.

[1] Morphological Characteristics

The methods described by Shirling and Gottlieb (Shirling, E. B. and D.Gottlieb: Methods for characterization of Streptomyces species.International Journal of Systematic Bacteriology, 16, 313-340, 1966)were employed principally for this taxonomic study.

Morphological observations were made with light and electron microscopeson cultures grown at 30° C. for 14 days on oatmeal agar, yeast-maltextract agar and inorganic salts-starch agar. The mature sporophoresformed Rectiflexibiles with 10 to 50 or more than 50 spores in eachchain. The spores were oblong or cylindrical, 0.5-0.7×0.7-0.8 μm in sizeby electron microscopic observation. Spore surfaces were smooth.

[2] Cultural Characteristics

Cultural characteristics were observed on ten kinds of media describedby Shirling and Gottlieb as mentioned above, and by Waksman (Waksman, S.A.: The actinomycetes, vol. 2: Classification, identification anddescription of genera and species. The Williams and Wilkins Co.,Baltimore, 1961).

The incubation was made at 30° C. for 14 days. The color names used inthis study were based on Guide to Color Standard (manual published byNippon Shikisai Kenkyusho, Tokyo). Colonies belonged to the gray colorseries when grown on oatmeal agar, yeast-malt extract agar and inorganicsalts-starch agar. Soluble pigment was produced in yeast-malt extractagar but not in other media. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cultural Characteristics of                                                     Strain No. 9993 and Streptomyces misakiensis IFO 12891                                  Cultural characteristics                                          Medium      No. 9993      IFO 12891                                           ______________________________________                                        Oatmeal Agar                                                                            G     Moderate      Moderate                                           A Gray Grayish White                                                          R Pale Pink Colorless                                                         S None None                                                                  Yeast-Malt G Moderate Moderate                                                Extract Agar A Light Gray Grayish White                                        R Dull Reddish Orange Light Brown                                             S Dull Reddish Orange None                                                   Inorganic Salts- G Moderate Moderate                                          Starch Agar A Pale Yellow Orange to Grayish White                               Light Gray                                                                   R Dark Orange Pale Yellowish Brown                                            S None None                                                                  Glucose- G Poor Moderate                                                      Asparagine A White Grayish White                                              Agar R Pale Brown Pale Yellowish Brown                                         S None Pale Brown                                                            Glycerin- G Moderate Moderate                                                 Asparagine A Pale Pink to White Grayish White                                 Agar R Pale Pink Pale Yellowish Brown                                          S None Pale Brown                                                            Czapek Agar G Poor Abundant                                                    A None Grayish White                                                          R Pale Pink Dark Orange to Dark                                                 Brown                                                                       S None None                                                                  Nutrient Agar G Poor Poor                                                      A White, Poor White                                                           R Colorless Colorless                                                         S None None                                                                  Potato-Dextrose G Poor Moderate                                               Agar A None Yellowish Gray                                                     R Pale Pink Brown                                                             S None None                                                                  Tyrosine Agar G Moderate Moderate                                              A White Grayish White to Light                                                  Gray                                                                        R Dull Reddish Orange Dark Orange to Black                                    S None None                                                                  Peptone-Yeast G Poor Poor                                                     Extract-Iron A None None                                                      Agar R Colorless Colorless                                                     S None None                                                                ______________________________________                                         Abbreviation: G = Growth, A = Aerial Mass Color, R = Reverse Side Color,      = Soluble Pigment,                                                       

The cell wall analysis was performed by the methods of Becker et al.(Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier:Rapid differentiation between Nocardia and Streptomyces by paperchromatography of whole cell hydrolysates: Appl. Microbiol., 12,421-423, 1966) and Yamaguchi (Yamaguchi, T.: Comparison of the cell wallcomposition of morphologically distinct-actinomycetes: J. Bacteriol.,89, 444-453, 1965). Analysis of whole cell hydrolysates of the strainNo. 9993 showed the presence of LL-diaminopimelic acid. Accordingly, thecell wall of this strain is believed to be of type I.

[3] Biological and Physiological Properties

Physiological properties of the strain No. 9993 were determinedaccording to the methods described by Shirling and Gottlieb as mentionedabove. The results are shown in Table 2. Temperature range and optimumtemperature for growth were determined on yeast-malt extract agar usinga temperature gradient incubator (made by Toyo Kagaku Sangyo Co., Ltd.).Temperature range for growth was from 18 to 35° C. with optimumtemperature at 28° C. Milk peptonization and gelatin liquefaction werepositive. Melanoid pigment production was negative.

                  TABLE 2                                                         ______________________________________                                        Physiological Properties of Strain No. 9993 and                                 Streptomyces misakiensis IFO 12891                                            Physiological properties                                                                        No. 9993    IFO 12891                                     ______________________________________                                        Temperature Range for Growth                                                                  18° C.-35° C.                                                               12° C.-35° C.                         Optimum Temperature 28° C. 28° C.                               Nitrate Reduction Negative Negative                                           Starch Hydrolysis Negative Positive                                           Milk Coagulation Negative Negative                                            Milk Peptonization Positive Weakly Positive                                   Melanin Production Negative Negative                                          Gelatin Liquefaction Positive Negative                                        H.sub.2 S Production Negative Negative                                        NaCl Tolerance (%) ≦3% 3%<, <5%                                      ______________________________________                                    

Utilization of carbon sources was examined according to the methods ofPridham and Gottlieb (Pridham, T. G. and D. Gottlieb: The utilization ofcarbon compounds by some Actinomycetales as an aid for speciesdetermination: J. Bacteriol., 56, 107-114, 1948). The growth wasobserved after 14 days incubation at 30° C.

Summarized carbon sources utilization of this strain is shown in Table3. Glycerin, maltose and sodium succinate could be utilized by thestrain No. 9993. Further, doubtful utilization of D-glucose, sucrose,D-mannose and salicin was also observed.

                  TABLE 3                                                         ______________________________________                                        Carbon Sources Utilization of Strain No. 9993 and                               Streptomyces misakiensis IFO 12691                                               Carbon Sources  No. 9993 IFO 12891                                       ______________________________________                                        D-Glucose        ±     -                                                     Sucrose ± -                                                                Glycerin + -                                                                  D-Xylose - -                                                                  D-Fructose - -                                                                Lactose - -                                                                   Maltose + -                                                                   Rhamnose - -                                                                  Raffinose - -                                                                 D-Galactose - +                                                               L-Arabinose - -                                                               D-Mannose ± -                                                              D-Trehalose - -                                                               Inositol - -                                                                  D-Mannitol - -                                                                Inulin - +                                                                    Cellulose - -                                                                 Salicin ± -                                                                Chitin - ±                                                                 Sodium Citrate - -                                                            Sodium Succinate + -                                                          Sodium Acetate - -                                                          ______________________________________                                         Symbols:                                                                      +: utilization                                                                ±: doubtful utilization                                                    -: no utilization                                                        

Microscopic studies and cell wall composition analysis of the strain No.9993 indicate that this strain belongs to the genus Streptomyces Waksmanand Henrici 1943.

Accordingly, a comparison of this strain was made with variousStreptomyces species in the light of the published descriptions[International Journal of Systematic Bacteriology, 18, 69 to 189, 279 to392 (1968) and 19, 391 to 512 (1969), and Bergy's Manual ofDeterminative Bacteriology 8th Edition (1974)].

As a result of the comparison, the strain No. 9993 is considered toresemble Streptomyces aburaviensis Nishimura et. al., Streptomycesavellaneus Baldacci and Grein and Streptomyces misakiensis Nakamura.Therefore, the cultural characteristics of the strain No. 9993 werecompared with the corresponding Streptomyces aburaviensis IFO 12830,Streptomyces avellaneus IFO 13451 and Streptomyces misakiensis IFO12891. As a result, the strain No. 9993 was the most similar toStreptomyces misakiensis IFO 12891. Therefore, the strain No. 9993 wasfurther compared with Streptomyces misakiensis IFO 12891 as shown in theabove Tables 1, 2 and 3. From further comparison, the strain No. 9993could be differentiated from Streptomyces misakiensis IFO 12891 in thefollowing points, and therefore the strain No. 9993 is considered to bea new species of Streptomyces and has been designated as Streptomycestsukubaensis sp. nov., referring to the soil, collected at Tsukuba-gun,from which the organism was isolated.

Difference from Streptomyces misakiensis IFO 12891

Cultural characteristics of the strain No. 9993 are different from theStreptomyces misakiensis IFO 12891 on oatmeal agar, yeast-malt extractagar, glucose-asparagine agar, Czapek agar and potato-dextrose agar.

Starch hydrolysis of the strain No. 9993 is negative, but that of theStreptomyces misakiensis IFO 12891 is positive.

Gelatin liquefaction of the strain No. 9993 is positive, but that of theStreptomyces misakiensis IFO 12891 is negative.

In carbon sources utilization, the strain No. 9993 can utilize glycerin,maltose and sodium succinate, but the Streptomyces misakiensis IFO 12891can not utilize them. And, the strain No. 9993 can not utilizeD-galactose and inulin, but the Streptomyces misakiensis IFO 12891 canutilize them.

PRODUCTION OF FR-900506, FR-900520 AND FR-900525 SUBSTANCES

The novel FR-900506, FR-900520 and FR-900525 substances of thisinvention can be produced by culturing a FR-900506, FR-900520 and/orFR-900525 substance(s)-producing strain belonging to the genusStreptomyces (e.g. Streptomyces tsukubaensis No. 9993, FERM BP-927) in anutrient medium.

In general, the FR-900506, FR-900520 and/or FR-900525 substance(s) canbe produced by culturing the FR-900506, FR-900520 and/or FR-900525substance(s)-producing strain in an aqueous nutrient medium containingsources of assimilable carbon and nitrogen, preferably under aerobicconditions (e.g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, xylose, galactose, glycerin, starch, dextrin, and thelike. Other sources which may be included are maltose, rhamnose,raffinose, arabinose, mannose, salicin, sodium succinate, and the like.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cottonseed meal, soybean meal, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder etc., as well as inorganic andorganic nitrogen compounds such as ammonium salts (e.g. ammoniumnitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acid,and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form, because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use. Whendesired, there may be added to the medium mineral salts such as sodiumor calcium carbonate, sodium or potassium phosphate, sodium or potassiumchloride, sodium or potassium iodide, magnesium salts, copper salts,cobalt salt and the like. If necessary, especially when the culturemedium foams seriously, a defoaming agent, such as liquid paraffin,fatty oil, plant oil, mineral oil or silicone may be added.

As the conditions for the production of the FR-900506, FR-900520 andFR-900525 substances in massive amounts, submerged aerobic culturalconditions are preferred therefor. For the production in small amounts,a shaking or surface culture in a flask or bottle is employed.Furthermore, when the growth is carried out in large tanks, it ispreferable to use the vegetative form of the organism for inoculation inthe production tanks in order to avoid growth lag in the process ofproduction of the FR-900506, FR-900520 and FR-900525 substances.Accordingly, it is desirable first to produce a vegetative inoculum ofthe organism by inoculating a relatively small quantity of culturemedium with spores or mycelia of the organism and culturing saidinoculated medium, and then to transfer the cultured vegetative inoculumaseptically to large tanks. The medium, in which the vegetative inoculumis produced, is substantially the same as or different from the mediumutilized for the production of the FR-900506, FR-900520 and FR-900525substances.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C. and 40° C., preferably 25-35° C., for a period of about 50 hours to150 hours, which may be varied according to fermentation conditions andscales.

Thus produced FR-900506, FR-900520 and/or FR-900525 substances can berecovered from the culture medium by conventional means which arecommonly used for the recovery of other known biologically activesubstances. The FR-900506, FR-900520 and FR-900525 substances producedare found in the cultured mycelium and filtrate, and accordingly theFR-900506, FR-900520 and FR-900525 substances can be isolated andpurified from the mycelium and the filtrate, which are obtained byfiltering or centrifuging the cultured broth, by a conventional methodsuch as concentration under reduced pressure, lyophilization, extractionwith a conventional solvent, pH adjustment, treatment with aconventional resin (e.g. anion or cation exchange resin, non-ionicadsorption resin, etc.), treatment with a conventional adsorbent (e.g.activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.),crystallization, recrystallization, and the like.

PHYSICAL AND CHEMICAL PROPERTIES OF FR-900506, FR-900520 AND FR-900525SUBSTANCES

The FR-900506, FR-900520 and FR-900525 substances produced according tothe aforementioned process possess the following physical and chemicalproperties.

FR-900506 Substance

(1) Form and Color:

white powder

(2) Elemental Analysis:

    ______________________________________                                        C:     64.72%,     H:    8.78%,   N:  1.59%                                      64.59%  8.74%  1.62%                                                       ______________________________________                                    

(3) Color Reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction, Ehrlichreaction, Dragendorff reaction and iodine vapor reaction

Negative: ferric chloride reaction, ninhydrin reaction and Molishreaction

(4) Solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethylether and benzene

Sparingly Soluble: hexane, petroleum ether

Insoluble: water

(5) Melting Point:

85-90° C.

(6) Specific Rotation:

[α]_(D) ²³ : -73° (c=0.8, CHCl₃)

(7) Ultraviolet Absorption Spectrum:

end absorption

(8) Infrared Absorption Spectrum:

ν_(max) ^(CHCl) 3: 3680, 3580, 3520, 2930, 2870, 2830, 1745, 1720, 1700,1645, 1450, 1380, 1350, 1330, 1310, 1285, 1170, 1135, 1090, 1050, 1030,1000, 990, 960(sh), 918 cm⁻¹

(9) ¹³ C Nuclear Magnetic Resonance Spectrum: ##EQU1## the chart ofwhich being shown in FIG. 1,

(10) ¹ H Nuclear Magnetic Resonance Spectrum:

the chart of which being shown in FIG. 2,

(11) Thin Layer Chromatography:

    ______________________________________                                        Stationary Phase                                                                          Developing Solvent                                                                              Rf Values                                       ______________________________________                                        silica gel plate                                                                          chloroform:methanol (10:1, v/v)                                                                 0.58                                               ethyl acetate 0.52                                                         ______________________________________                                    

(12) Property of the Substance:

neutral substance

With regard to the FR-900506 substance, it is to be noted that in caseof measurements of ¹³ C and ¹ H nuclear magnetic resonance spectra, thissubstance showed pairs of the signals in various chemical shifts.

The FR-900506 substance thus characterized further possesses thefollowing properties.

(i) The measurements of ¹³ C Nuclear Magnetic Resonance Spectra at 25°C. and 60° C. revealed the fact that the intensities of each pair of thevarious signals therein were changed.

(ii) The measurements of the thin layer chromatography and the highperformance liquid chromatography revealed that the FR-900506 substanceoccurs as a single spot in the thin layer chromatography and a singlepeak in the high performance liquid chromatography, respectively.

This white powder of the FR-900506 substance could be transformed into aform of crystals by recrystallization thereof from acetonitrile, whichpossess the following physical and chemical properties.

(1) Form and Color:

colorless prisms

(2) Elemental Analysis:

C: 64.30%, H: 8.92%, N: 1.77% 64.20%, 8.86%, 1.72%,

(3) Melting Point:

127-129° C.

(4) Specific Rotation:

[α]_(D) ²³ : -84.4° (c=1.02, CHCl₃)

(5) ¹³ C Nuclear Magnetic Resonance Spectrum: ##EQU2## the chart ofwhich being shown in FIG. 3,

(6) ¹ H Nuclear Magnetic Resonance Spectrum:

the chart of which being shown in FIG. 4.

Other physical and chemical properties, that is, the color reaction,solubility, ultraviolet absorption spectrum, infrared absorptionspectrum, thin layer chromatography and property of the substance of thecolorless prisms of the FR-900506 substance were the same as those forthe white powder of the same under the identical conditions.

From the above physical and chemical properties and the analysis of theX ray diffraction, the FR-900506 substance could be determined to havethe following chemical structure. ##STR4##17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone

FR-900520 Substance

The physical and chemical properties are mentioned later.

FR-900525 Substance

(1) Form and Color:

white powder

(2) Elemental Analysis:

C: 65.17%, H: 8.53%, N: 1.76%

(3) Color Reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction, Ehrlichreaction, Dragendorff reaction and iodine vapor reaction

Negative: ferric chloride reaction, ninhydrin reaction and Molishreaction

(4) Solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethylether and benzene

Sparingly Soluble: hexane, petroleum ether

Insoluble: water

(5) Melting Point:

85-89° C.

(6) Specific Rotation:

[α]_(D) ²³ : -88° (c=1.0, CHCl₃)

(7) Ultraviolet Absorption Spectrum:

end absorption

(8) Infrared Absorption Spectrum:

ν_(max) ^(CHCl) 3: 3680, 3580, 3475, 3340, 2940, 2880, 2830, 1755, 1705,1635, 1455, 1382, 1370, 1330, 1310, 1273, 1170, 1135, 1093, 1050, 1020,995, 970, 920, 867 cm⁻¹

(9) ¹³ C Nuclear Magnetic Resonance Spectrum: ##EQU3## the chart ofwhich being shown in FIG. 5,

(10) ¹ H Nuclear Magnetic Resonance Spectrum:

the chart of which being shown in FIG. 6,

(11) Thin Layer Chromatography:

Developing Stationary Phase Solvent Rf Value silica gel plate ethylacetate 0.34

(12) Property of the Substance:

neutral substance

With regard to the FR-900525 substance, it is to be noted that in caseof measurements of ¹³ C and ¹ H nuclear magnetic resonance spectra, thissubstance showed pairs of the signals in various chemical shifts,however, in case of measurements of the thin layer chromatography andthe high performance liquid chromatography, the FR-900525 substanceshowed a single spot in the thin layer chromatography and a single peakin the high performance liquid chromatography, respectively.

From the above physical and chemical properties and the success of thedetermination of the chemical structure of the FR-900506 substance, theFR-900525 substance could be determined to have the following chemicalstructure. ##STR5##16-Allyl-1,13-dihydroxy-11-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-22,24-dimethoxy-12,18,20,26-tetramethyl-10,27-dioxa-4-azatricyclo-[21.3.1.0⁴,8]heptacos-17-ene-2,3,9,15-tetraone

[B] The FR-900520 and FR-900523 substances of this invention can beproduced by fermentation of FR-900520 and/or FR-900523substance(s)-producing strain belonging to the genus Streptomyces suchas Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 in anutrient medium.

The Microorganism

The microorganism which can be used for the production of the FR-900520and/or FR-900523 substances is FR-900520 and/or FR-900523substance(s)-producing strain belonging to the genus Streptomyces, amongwhich Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 has beennewly isolated from a soil sample collected at Yakushima, KagoshimaPrefecture, Japan.

A lyophilized sample of the newly isolated Streptomyces hygroscopicussubsp. yakushimaensis No. 7238 has been deposited with the FermentationResearch Institute, Agency of Industrial Science and Technology (No.1-3,Higashi 1-chome, Yatabemachi, Tsukuba-gun, Ibaraki Prefecture, Japan)under the number of FERM P-8043 (deposited date: Jan. 12th, 1985), andthen converted to Budapest Treaty route of the same depository on Oct.19, 1985 under the new deposit number of FERM BP-928.

It is to be understood that the production of the novel FR-900520 andFR-900523 substances is not limited to the use of the particularorganism described herein, which is given for the illustrative purposeonly. This invention also includes the use of any mutants which arecapable of producing the FR-900520 and/or FR-900523 substance(s)including natural mutants as well as artificial mutants which can beproduced from the described organism by conventional means such asirradiation of X-rays, ultra-violet radiation, treatment withN-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.

The Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 has thefollowing morphological, cultural, biological and physiologicalcharacteristics.

[1] Morphological Characteristics

The methods described by Shirling and Gottlieb (Shirling, E. B. and D.Gottlieb: Methods for characterization of Streptomyces species.International Journal of Systematic Bacteriology, 16, 313-340, 1966)were employed principally for this taxonomic study.

Morphological observations were made with light and electron microscopeson cultures grown at 30° C. for 14 days on oatmeal agar, yeast-maltextract agar and inorganic salts-starch agar. The mature sporophoreswere moderately short and formed Retinaculiaperti and Spirales withabout 20 spores in each chain. Hygroscopic spore mass were seen in theaerial mycelia on oatmeal agar and inorganic salts-starch agar. Surfaceirregularities on spores were intermediate between very short, thickspines and warts.

[2] Cultural Characteristics

Cultural characteristics were observed on ten kinds of media describedby Shirling and Gottlieb as mentioned above, and by Waksman (Waksman, S.A.: The actinomycetes, vol. 2: Classification, identification anddescription of genera and species. The Williams and Wilkins Co.,Baltimore, 1961).

The incubation was made at 30° C. for 14 days. The color names used inthis study were based on Guide to Color Standard (manual published byNippon Shikisai Kenkyusho, Tokyo). Colonies belonged to the gray colorseries when grown on oatmeal agar, yeast-malt extract agar and inorganicsalts-starch agar. Soluble pigment was not produced in the examinedmedia. The results are shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Cultural Characteristics of Strain No. 7238, Streptomyces antimycoticus       IFO 12839                                                                       and Streptomyces hygroscopicus subsp. glebosus IFO 13786                              Cultural Characteristics                                            Medium    No. 7238  IFO 12839 IFO 13786                                       __________________________________________________________________________    Oatmeal Agar                                                                          G Poor      Poor      Poor                                               A Grayish Yellow Brown Grayish Yellow Brown Grayish Yellow Brown                                           R Pale Yellow Pale Yellow Pale Yellow                                         S None None None                                Yeast-Malt G Moderate Abundant Moderate                                       Extract Agar A Grayish White Gray Gray                                         P Pale Yellowish Brown Pale Yellowish Brown Dark Orange                       S None None None                                                             Inorganic Salts- G Moderate Moderate Moderate                                 Starch Agar A Gray to Black Gray Light Gray                                    R Pale Yellow orange Yellowish Gray Pale Yellow Orange                        S None None None                                                             Glucose- G Moderate Moderate Moderate                                         Asparagine A Grayish White Gray White                                         Agar R Pale Yellow Orange Pale Yellow Orange Pale Yellow Orange                                             S None None None                                Glycerin- G Moderate Moderate Moderate                                        Asparagine A White Gray Light Gray                                            Agar R Yellowish Gray Yellowish Gray Grayish Yellow Brown                      S None None None                                                             Czapek Agar G Moderate Moderate Moderate                                       A Grayish White Grayish White White                                           R Pale Yellowish Brown Pale Yellowish Brown Pale Yellowish Brown                                           S None None None                                Nutrient Agar G Moderate Moderate Moderate                                     A Grayish White Gravish White None                                            p Pale Yellow Pale Yellow Pale Yellow                                         S None None None                                                             Potato-Dextrose G Moderate Moderate Moderate                                  Agar A White, Poor Pale Reddish Brown Pale Pink to White                       R Pale Yellow Orange Pale Yellow Orange Pale Yellowish Brown                  S None None None                                                             Tyrosine Agar G Moderate Moderate Moderate                                     A White Grayish White Gray to Black                                           R Pale Yellowish Brown Brown Pale Yellowish Brown                             S None Brown None                                                            Peptone-Yeast G Moderate Moderate Moderate                                    Extract-Iron A None Grayish White None                                        Agar R Pale Yellow Pale Yellow Colorless                                       S None None None                                                           __________________________________________________________________________     Abbreviation: G = Growth, A = Aerial Mass Color, R = Reverse Side Color,      = Soluble Pigment,                                                       

The cell wall analysis was performed by the methods of Becker et al.(Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier:Rapid differentiation between Nocardia and Streptomyces by paperchromatography of whole cell hydrolysates: Appl. Microbiol., 12,421-423, 1964) and Yamaguchi (Yamaguchi, T.: Comparison of the cell wallcomposition of morphologically distinct actinomycetes: J. Bacteriol.,89, 444-453, 1965). Analysis of whole cell hydrolysates of the strainNo. 7238 showed the presence of LL-diaminopimelic acid. Accordingly, thecell wall of this strain is believed to be of type I.

[3] Biological and Physiological Properties

Physiological properties of the strain No. 7238 were determinedaccording to the methods described by Shirling and Gottlieb as mentionedabove. The results are shown in Table 5. Temperature range and optimumtemperature for growth were determined on yeast-malt extract agar usinga temperature gradient incubator (made by Toyo Kagaku Sangyo Co., Ltd.).Temperature range for growth was from 18 to 36° C. with optimumtemperature at 28° C. Starch hydrolysis and gelatin liquefaction werepositive. No melanoid pigment was produced.

                  TABLE 5                                                         ______________________________________                                        Physiological Properties of Strain No. 7238, Streptomyces antimycoticus        IFO 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13786             Physiological                                                                                                    properties No. 7238 IFO 12839 IFO                                            13786                                     ______________________________________                                        Temperature Range                                                                         18° C.-36° C.                                                              18° C.-38° C.                                                              16° C.-35° C.                   for Growth                                                                    Optimum Temperature 28° C. 28° C. 27° C.                 Nitrate Reduction Negative Negative Negative                                  Starch Hydrolysis Positive Positive Positive                                  Milk Coagulation Negative Negative Negative                                   Milk Peptonization Negative Negative Positive                                 Melanin Production Negative Negative Negative                                 Gelatin Liquefaction Positive Positive Positive                               H.sub.2 S Production Negative Negative Negative                               Urease Activity Negative Negative Negative                                    NaCl Tolerance (%) 7%<, <10% 7%<, <10% 5%<, <7%                             ______________________________________                                    

Utilization of carbon sources was examined according to the methods ofPridham and Gottlieb (Pridham, T. G. and D. Gottlieb: The utilization ofcarbon compounds by some Actinomycetales as an aid for speciesdetermination: J. Bacteriol., 56, 107-114, 1948). The growth wasobserved after 14 days incubation at 30° C.

Summarized carbon sources utilization of this strain is shown in Table6. D-Glucose, sucrose, lactose, maltose, D-trehalose, inositol, inulinand salicin could be utilized by the strain No. 7238.

                  TABLE 6                                                         ______________________________________                                        Carbon Sources Utilization of Strain No. 7238,                                  Streptomyces antimycoticus IFO 12839 and                                      Streptomyces hygroscopicus subsp. glebosus IFO 13786                            Carbon Sources                                                                            No. 7238    IFO 12839                                                                            IFO 13786                                  ______________________________________                                        D-Glucose   +           +        +                                              Sucrose + + +                                                                 Glycerin - + +                                                                D-Xylose - ± +                                                             D-Fructose - + +                                                              Lactose + + -                                                                 Maltose + - +                                                                 Rhamnose - + -                                                                Raffinose - + +                                                               D-Galactose - + +                                                             L-Arabinose - ± ±                                                       D-Mannose - + +                                                               D-Trehalose + ± +                                                          Inositol + + +                                                                D-Mannitol - + +                                                              Inulin + + -                                                                  Cellulose ± - -                                                            Salicin + + -                                                                 Chitin ± - -                                                               Sodium Citrate - - ±                                                       Sodium Succinate - + +                                                        Sodium Acetate - - -                                                        ______________________________________                                         Symbols:                                                                      +: utilization                                                                ±: doubtful utilization                                                    -: no utilization                                                        

Microscopic studies and cell wall composition analysis of the strain No.7238 indicate that this strain belongs to the genus Streptomyces Waksmanand Henrici 1943.

Accordingly, a comparison of this strain was made with variousStreptomyces species in the light of the published descriptions[International Journal of Systematic Bacteriology, 18, 69 to 189, 279 to392 (1968) and 19, 391 to 512 (1969), and Bergy's Manual ofDeterminative Bacteriology 8th Edition (1974)].

As a result of the comparison, the strain No. 7238 is considered toresemble Streptomyces antimycoticus Waksman 1957 and Streptomyceshygroscopicus subsp. glebosus Ohmori, et. al. 1962. Therefore, thecultural characteristics of the strain No. 7238 were further comparedwith the corresponding Streptomyces antimycoticus IFO 12839 andStreptomyces hygroscopicus subsp. glebosus IFO 13786 as shown in theabove Tables 4, 5 and 6. From further comparison, the strain No. 7238could be differentiated from Streptomyces antimycoticus IFO 12839 andStreptomyces hygroscopicus subsp. glebosus IFO 13786 in the followingpoints.

(i) Difference from Streptomyces antimycoticus IFO 12839

Cultural characteristics of the strain No. 7238 are different from theStreptomyces antimycoticus IFO 12839 on yeast-malt extract agar,glucose-asparagine agar, glycerin-asparagine agar, potato-dextrose agarand tyrosine agar.

In carbon sources utilization, the strain No. 7238 can utilize maltose,but the Streptomyces antimycoticus IFO 12839 can not utilize it. And,the strain No. 7238 can not utilize glycerin, D-fructose, rhamnose,raffinose, D-galactose, D-mannose, mannitol and sodium succinate, butthe Streptomyces antimycoticus IFO 12839 can utilize them.

(ii) Difference from Streptomyces hygroscopicus subsp. glebosus IFO13786

Cultural characteristics of the strain No. 7238 are different from theStreptomyces hygroscopicus subsp. glebosus IFO 13786 on yeast-maltextract agar, potato-dextrose agar and tyrosine agar.

Milk peptonization of the strain No. 7238 is negative, but that of theStreptomyces hygroscopicus subsp. glebosus IFO 13786 is positive. Thestrain No. 7238 can grow in the presence of 7% NaCl, but theStreptomyces hygroscopicus subsp. glebosus IFO 13786 can not grow underthe same condition.

In carbon sources utilization, the strain No. 7238 can utilize lactose,inulin and salicin, but the Streptomyces hygroscopicus subsp. glebosusIFO 13786 can not utilize them. And, the strain No. 7238 can not utilizeglycerin, D-xylose, D-fructose, raffinose, D-galactose, D-mannose,mannitol and sodium succinate, but the Streptomyces hygroscopicus subsp.glebosus IFO 13786 can utilize them.

However, the strain No. 7238 forms hygroscopic spore mass in the aerialmycelia on oatmeal agar and inorganic salts-starch agar, and furthermorphological and cultural characteristics of the strain No. 7238 aresimilar to the Streptomyces hygroscopicus subsp. glebosus IFO 13786.Therefore, the strain No. 7238 is considered to belong to Streptomyceshygroscopicus, but the strain No. 7238 is different from theStreptomyces hygroscopicus subsp. glebosus IFO 13786, though this knownstrain is the most similar to the strain No. 7238 in Streptomyceshygroscopicus subspecies. From the above facts, the strain No. 7238 isconsidered to be a new subspecies of Streptomyces hygroscopicus and hasbeen designated as Streptomyces hygroscopicus subsp. yakushimaensissubsp. nov., referring to the soil collected at Yakushima, from whichthe organism was isolated.

PRODUCTION OF FR-900520 and FR-900523 SUBSTANCES

The novel FR-900520 and/or FR-900523 substance(s) can be produced byculturing FR-900520 and/or FR-900523 substance(s)-producing strainbelonging to the genus Streptomyces (e.g. Streptomyces hygroscopicussubsp. yakushimaensis No. 7238, FERM BP-928) in a nutrient medium.

In general, the FR-900520 and/or FR-900523 substance(s) can be producedby culturing the FR-900520 and/or FR-900523 substance(s)-producingstrain in an aqueous nutrient medium containing sources of assimilablecarbon and nitrogen, preferably under aerobic conditions (e.g. shakingculture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, sucrose, lactose, glycerin, starch, dextrin, and thelike. Other sources which may be included are maltose, D-trehalose,inositol, inulin, salicin, and the like.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cottonseed meal, soybean meal, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder etc., as well as inorganic andorganic nitrogen compounds such as ammonium salts (e.g. ammoniumnitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acid,and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form, because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use. Whendesired, there may be added to the medium mineral salts such as sodiumor calcium carbonate, sodium or potassium phosphate, sodium or potassiumchloride, sodium or potassium iodide, magnesium salts, copper salts,cobalt salt and the like. If necessary, especially when the culturemedium foams seriously, a defoaming agent, such as liquid paraffin,fatty oil, plant oil, mineral oil or silicone may be added.

As the conditions for the production of the FR-900520 and FR-900523substances in massive amounts, submerged aerobic cultural conditions arepreferred therefor. For the production in small amounts, a shaking orsurface culture in a flask or bottle is employed. Furthermore, when thegrowth is carried out in large tanks, it is preferable to use thevegetative form of the organism for inoculation in the production tanksin order to avoid growth lag in the process of production of theFR-900520 and FR-900523 substances. Accordingly, it is desirable firstto produce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with spores or mycelia ofthe organism and culturing said inoculated medium, and then to transferthe cultured vegetative inoculum aseptically to large tanks. The medium,in which the vegetative inoculum is produced, is substantially the sameas or different from the medium utilized for the production of theFR-900520 and FR-900523 substances.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C. and 40° C., preferably 25-35° C., for a period of about 50 hours to150 hours, which may be varied according to fermentation conditions andscales.

Thus produced FR-900520 and/or FR-900523 substance(s) can be recoveredfrom the culture medium by conventional means which are commonly usedfor the recovery of other known biologically active substances. TheFR-900520 and is FR-900523 substances produced are mainly found in thecultured mycelium, and accordingly the FR-900520 and FR-900523substances can be isolated and purified from the mycelium, which areobtained by filtering or centrifuging the cultured broth, by aconventional method such as concentration under reduced pressure,lyophilization, extraction with a conventional solvent, pH adjustment,treatment with a conventional resin (e.g. anion or cation exchangeresin, non-ionic adsorption resin, etc.), treatment with a conventionaladsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose,alumina, etc.), crystallization, recrystallization, and the like.

Particularly, the FR-900520 substance and the FR-900523 substance can beseparated by dissolving the materials containing both products producedby fermentation in an appropriate solvent such as ethyl acetate,n-hexane, and the like, and then by subjecting said solution tochromatography, for example, on silica gel in a column with anappropriate organic solvent such as ethyl acetate and n-hexane, or amixture thereof. And each of the FR-900520 substance and the FR-900523substance thus separated can be further purified by a conventionalmethod, for example, recrystallization, re-chromatography, highperformance liquid chromatography, and the like.

PHYSICAL AND CHEMICAL PROPERTIES OF FR-900520 and FR-900523 SUBSTANCES

FR-900520 Substance

(1) Form and Color:

colorless plates

(2) Elemental Analysis:

C: 64.81%, H: 8.82%, N: 1.55%

(3) Color Reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction, Ehrlichreaction, Dragendorff reaction and iodine vapor reaction

Negative: ferric chloride reaction, ninhydrin reaction and Molishreaction

(4) Solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethylether and benzene

Sparingly Soluble: n-hexane, petroleum ether

Insoluble: water

(5) Melting Point:

163-165° C.

(6) Specific Rotation:

[α]_(D) ²³ : -84.1° (c=1.0, CHCl₃)

(7) Ultraviolet Absorption Spectrum:

end absorption

(8) Infrared Absorption Spectrum:

ν_(max) ^(CHCl) 3: 3680, 3575, 3520, 2940, 2875, 2825, 1745, 1725, 1700,1647, 1610(sh), 1452, 1380, 1350, 1330, 1285, 1170, 1135, 1090, 1030,1005, 990, 980(sh), 960(sh), 913, 908(sh) cm⁻¹

(9) ¹³ C Nuclear Magnetic Resonance Spectrum: ##EQU4## the chart ofwhich being shown in FIG. 7,

(10) ¹ H Nuclear Magnetic Resonance Spectrum:

the chart of which being shown in FIG. 8,

(11) Thin Layer Chromatography:

Developing Stationary Phase Solvent Rf Values chloroform silica gelplate: methanol (20:1, v/v) 0.38 ethyl acetate 0.51

(12) Property of the Substance:

neutral substance

With regard to the FR-900520 substance, it is to be noted that in caseof measurements of ¹³ C and ¹ H nuclear magnetic resonance spectra, thissubstance shows pairs of the signals in various chemical shifts,however, in case of measurements of the thin layer chromatography andthe high performance liquid chromatography, the FR-900520 substanceshowed a single spot in the thin layer chromatography and a single peakin the high performance liquid chromatography, respectively.

From the above physical and chemical properties and the success of thedetermination of the chemical structure of the FR-900506 substance, theFR-900520 substance could be determined to have the following chemicalstructure. ##STR6##17-Ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone

FR-900523 Substance

(1) Form and Color:

colorless needles

(2) Elemental Analysis:

C: 64.57%, H: 8.84%, N: 1.81%

(3) Color Reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction, Ehrlichreaction, Dragendorff reaction and iodine vapor reaction

Negative: ferric chloride reaction and ninhydrin reaction

(4) Solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethylether and benzene

Sparingly Soluble: n-hexane and petroleum ether

Insoluble: water

(5) Melting Point:

152-154° C.

(6) Specific Rotation:

[α]_(D) ²³ : -73.0° (C=0.65, CHCl₃)

(7) Ultraviolet Absorption Spectrum:

end absorption

(8) Infrared Absorption Spectrum:

ν_(max) ^(CHCl) 3: 3670, 3580, 3510, 2930, 2875, 2825, 1745, 1722, 1700,1647, 1450, 1380, 1350, 1330, 1307, 1285, 1170, 1135, 1090, 1050, 1030,1000, 990, 978, 960, 930, 915, 888, 870, 850 cm⁻¹

(9) ¹³ C Nuclear Magnetic Resonance Spectrum: ##EQU5## the chart ofwhich being shown in FIG. 9,

(10) ¹ H Nuclear Magnetic Resonance Spectrum:

the chart of which being shown in FIG. 10,

(11) Thin Layer Chromatography:

Developing Stationary Phase Solvent Rf Values chloroform methanol 0.38silica gel plate (20:1, v/v) ethyl acetate 0.51

(12) Property of the Substance:

neutral substance

With regard to the FR-900523 substance, it is to be noted that in caseof measurements of ¹³ C and ¹ H nuclear magnetic resonance spectra, thissubstance shows pairs of the signals in various chemical shifts,however, in case of measurements of the thin layer chromatography andthe high performance liquid chromatography, the FR-900523 substanceshowed a single spot in the thin layer chromatography and a single peakin the high performance liquid chromatography, respectively.

From the above physical and chemical properties and the success of thedetermination of the chemical structure of the FR-900506 substance, theFR-900523 substance could be determined to have the following chemicalstructure. ##STR7##1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,17,21,27-pentamethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone

[II] Synthetic Processes

(1) Process 1: (Introduction of Hydroxy-Protective Group)

The compound (Ib) or a salt thereof can be prepared by introducing ahydroxy-protective group into the compound (Ia) or a salt thereof.

Suitable introducing agent of the hydroxy-protective group used in thisreaction may be a conventional one such as di(lower)alkyl sulfoxide, forexample, lower alkyl methyl sulfoxide (e.g. dimethyl sulfoxide, ethylmethyl sulfoxide, propyl methyl sulfoxide, isopropyl methyl sulfoxide,butyl methyl sulfoxide, isobutyl methyl sulfoxide, hexyl methylsulfoxide, etc.), trisubstituted silyl compound such astri(lower)alkylsilyl halide (e.g. trimethylsilyl chloride, triethylsilylbromide, tributylsilyl chloride, tert-butyl-dimethylsilyl chloride,etc.), lower alkyl-diarylsilyl halide (e.g. methyl-diphenylsilylchloride, ethyl-diphenylsilyl bromide, propyl-ditolylsilyl chloride,tert-butyl-diphenylsilyl chloride, etc.), and acylating agent which iscapable of introducing the acyl group as mentioned before such ascarboxylic acid, sulfonic acid, carbamic acid and their reactivederivative, for example, an acid halide, an acid anhydride, an activatedamide, an activated ester, isocyanate, and the like. Preferable exampleof such reactive derivative may include acid chloride, acid bromide, amixed acid anhydride with an acid such as substituted phosphoric acid(e.g. dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoricacid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.),dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuricacid, alkyl carbonate (e.g. methyl carbonate, ethyl carbonate, propylcarbonate, etc.), aliphatic carboxylic acid (e.g. pivalic acid,pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetictrifluoroacetic acid, etc.), aromatic carboxylic acid (e.g. benzoicacid, etc.), a symmetrical acid anhydride, an activated acid amide witha heterocyclic compound containing imino function such as imidazole,4-substituted imidazole, dimethylpyrazole, triazole and tetrazole, anactivated ester (e.g. p-nitrophenyl ester, 2,4-dinitrophenyl ester,trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester,phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioester,p-cresyl thioester, carboxymethyl thioester, pyridyl ester, piperidinylester, 8-quinolyl thioester, or an ester with a N-hydroxy compound suchas N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole,1-hydroxy-6-chlorobenzotriazole, etc.), isocyanate, and the like.

In this reaction, in case that the di(lower)alkyl sulfoxide is used asan introducing agent of the hydroxy-protective group, the reaction isusually conducted in the presence of lower alkanoic anhydride such asacetic anhydride.

Further, in case that the trisubstituted silyl compound is used as anintroducing agent of the hydroxy-protective group, the reaction ispreferable conducted in the presence of a conventional condensing agentsuch as imidazole, and the like.

Still further, in case that the acylating agent is used as anintroducing agent of the hydroxy-protective group, the reaction ispreferably conducted in the presence of an organic or inorganic basesuch as alkali metal (e.g. lithium, sodium, potassium, etc.), alkalineearth metal (e.g. calcium, etc.), alkali metal hydride (e.g. sodiumhydride, etc.), alkaline earth metal hydride (e.g. calcium hydride,etc.), alkali metal hydroxide (e.g. sodium hydroxide, potassiumhydroxide, etc.), alkali metal carbonate (e.g. sodium carbonate,potassium carbonate, etc.), alkali metal hydrogen carbonate (e.g. sodiumhydrogen carbonate, potassium hydrogen carbonate, etc.), alkali metalalkoxide (e.g. sodium methoxide, sodium ethoxide, potassiumtert-butoxide, etc.), alkali metal alkanoic acid (e.g. sodium acetate,etc.), trialkylamine (e.g. triethylamine, etc.), pyridine compound (e.g.pyridine, lutidine, picoline, 4-N,N-dimethylaminopyridine, etc.),quinoline, and the like.

In case that the acylating agent is used in a free form or its salt inthis reaction, the reaction is preferably conducted in the presence of aconventional condensing agent such as a carbodiimide compound [e.g.N,N'-dicyclohexylcarbodiimide,N-cyclohexyl-N'-(4-diethylaminocyclohexyl)carbodiimide,N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide,N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, etc.], a keteniminecompound (e.g. N,N'-carbonylbis(2-methylimidazole),pentamethyleneketene-N-cyclohexylimine,diphenylketene-N-cyclohexylimine, etc.); an olefinic or acetylenic ethercompounds (e.g. ethoxyacetylene, β-cyclovinylethyl ether), a sulfonicacid ester of N-hydroxybenzotriazole derivative [e.g.1-(4-chlorobenzenesulfonyloxy)6-chloro-1H-benzotriazole, etc.], and thelike.

The reaction is usually conducted in a conventional solvent which doesnot adversely influence the reaction such as water, acetone,dichloromethane, alcohol (e.g. methanol, ethanol, etc.),tetrahydrofuran, pyridine, benzene, N,N-dimethylformamide, etc., or amixture thereof, and further in case that the base or the introducingagent of the hydroxy-protective group is in liquid, it can also be usedas a solvent.

The reaction temperature is not critical and the reaction is usuallyconducted under from cooling to heating.

This process includes, within a scope thereof, a case that during thereaction, the hydroxy group for R² of the compound (Ia) may occasionallybe transformed into the corresponding protected hydroxy group in theobject compound (Ib).

Further, this process also includes, within a scope thereof, a case thatwhen the di(lower)alkyl sulfoxide is used as an introducing agent of thehydroxy-protective group in the presence of lower alkanoic anhydride,the compound (Ia) having a partial structure of the formula: ##STR8##wherein R² is hydroxy, may occasionally be oxidized during the reactionto give the compound (Ib) having a partial structure of the formula##STR9## wherein R² is hydroxy. (2) Process 2: (Introduction ofHydroxy-Protective Group)

The compound (Id) or a salt thereof can be prepared by introducing ahydroxy-protective group into the compound (Ic) or a salt thereof.

The reaction can be conducted by substantially the same method as thatof Process 1, and therefore the reaction conditions (e.g. base,condensing agent, solvent, reaction temperature, etc.) are referred tothose of Process 1.

This process includes, within a scope thereof, a case that during thereaction, the hydroxy group for R¹ of the compound (Ic) may frequentlybe transformed into the corresponding protected hydroxy group in theobject compound (Id).

(3) Process 3: (Formation of Double Bond)

The compound (If) or a salt thereof can be prepared by reacting thecompound (Ie) or a salt thereof with a base.

Suitable base to be used in this reaction may include one as exemplifiedin Process 1.

This reaction can also be conducted by reacting the compound (Ie), whereR² is hydroxy, with an acylating agent in the presence of a base.

The reaction is usually conducted in a conventional solvent which doesnot adversely influence the reaction such as water, acetone,dichloromethane, alcohol (e.g. methanol, ethanol, propanol, etc.),tetrahydrofuran, pyridine, N,N-dimethylformamide, etc., or a mixturethereof, and further in case that the base is in liquid, it can also beused as a solvent.

The reaction temperature is not critical and the reaction is usuallyconducted under from cooling to heating.

(4) Process 4: (Oxidation of Hydroxyethylene Group)

The compound (Ih) or a salt thereof can be prepared by oxidizing thecompound (Ig) or a salt thereof.

The oxidizing agent to be used in this reaction may includedi(lower)alkyl sulfoxide such as those given in Process 1.

This reaction is usually conducted in the presence of lower alkanoicanhydride such as acetic anhydride in a conventional solvent which doesnot adversely influence the reaction such as acetone, dichloromethane,ethyl acetate, tetrahydrofuran, pyridine, N,N-dimethylformamide, etc.,or a mixture thereof, and further in case that the lower alkanoicanhydride is in liquid, it can also be used as a solvent.

The reaction temperature is not critical and the reaction is usuallyconducted under from cooling to heating.

This process includes, within a scope thereof, a case that during thereaction the hydroxy group for R¹ of the starting compound (Ig) mayoccasionally be transformed into 1-(lower alkylthio)(lower)alkyloxygroup in the object compound (Ih).

(5) Process 5:(Reduction of Allyl Group)

The compound (Ij) or a salt thereof can be obtained by reducing thecompound (Ii) or a salt thereof.

Reduction in this process can be conducted by a conventional methodwhich is capable of reducing an allyl group to a propyl group, such ascatalytic reduction, or the like.

Suitable catalysts used in catalytic reduction are conventional onessuch as platinum catalysts (e.g. platinum plate, spongy platinum,platinum black, colloidal platinum, platinum oxide, platinum wire,etc.), palladium catalysts (e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.), nickel catalysts(e.g. reduced nickel, nickel oxide, Raney nickel, etc.), cobaltcatalysts (e.g. reduced cobalt, Raney cobalt, etc.), iron catalysts(e.g. reduced iron, Raney iron, etc.), copper catalysts (e.g. reducedcopper, Raney copper, Ullman copper, etc.), and the like.

The reduction is usually conducted in a conventional solvent which doesnot adversely influence the reaction such as water, methanol, ethanol,propanol, pyridine, ethyl acetate, N,N-dimethylformamide,dichloromethane, or a mixture thereof.

The reaction temperature of this reduction is not critical and thereaction is usually conducted under from cooling to warming.

(6) Process 6: (Removal of the carboxy-protective group)

The compound (Il) or a salt thereof can be prepared by removing thecarboxy-protective group from the compound (Ik) or a salt thereof.

The removal reaction in this process can be conducted in a conventionalmanner which is capable of transforming atri(lower)alkylsilyl(lower)alkoxycarbonyl group to a carboxy group, thatis, in the presence of tetra(lower)alkylammonium fluoride (e.g.tetrabutylammonium fluoride, etc.), potassium fluoride, hydrogenfluoride, and the like.

This reaction is usually conducted in a conventional solvent which doesnot adversely influence the reaction such as tetrahydrofuran, and thelike.

The reaction temperature is not critical and the reaction is usuallyconducted under from cooling to warming.

The object tricyclo compounds (I) obtained according to the syntheticprocesses 1 to 6 as explained above can be isolated and purified in aconventional manner, for example, extraction, precipitation, fractionalcrystallization, recrystallization, chromatography, and the like.

Suitable salts of the compounds (I) and (Ia) to (Il) may includepharmaceutically acceptable salts such as basic salts, for example,alkali metal salt (e.g. sodium salt, potassium salt, etc.), alkalineearth metal salt (e.g. calcium salt, magnesium salt, etc.), ammoniumsalt, amine salt (e.g. triethylamine salt, N-benzyl-N-methylamine salt,etc.) and other conventional organic salts.

It is to be noted that in the aforementioned reactions in the syntheticprocesses 1 to 6 or the post-treatment of the reaction mixture therein,the conformer and/or stereo isomer(s) due to asymmetric carbon atom(s)or double bond(s) of the starting and object compounds may occasionallybe transformed into the other conformer and/or stereoisomer(s), and suchcases are also included within the scope of the present invention.

The tricyclo compounds (I) of the present invention possesspharmacological activities such as immunosuppressive activity,antimicrobial activity, and the like, and therefore are useful for thetreatment and prevention of the resistance by transplantation of organsor tissues such as heart, kidney, liver, medulla ossium, skin, etc.,graft-versus-host diseases by medulla ossium transplantation, autoimmunediseases such as rheumatoid arthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, etc., infectious diseases caused by pathogenicmicroorganisms, and the like.

As examples for showing such pharmacological activities, somepharmacological test data of the tricyclo compounds are illustrated inthe following.

Test 1

Suppression of Tricyclo Compounds (I) in in vitro Mixed LymphocyteReaction (MLR)

The MLR test was performed in microtiter plates, with each wellcontaining 5×10⁵ C57BL/6 responder cells (H-2^(b)), 5×10⁵ mitomycin Ctreated (25 μg/ml mitomycin C at 37° C. for 30 minutes and washed threetimes with RPMI 1640 medium) BALB/C stimulator cells (H-2^(d)) in 0.2 mlRPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM sodiumhydrogen carbonate, penicillin (50 unit/ml) and streptomycin (50 μg/ml).The cells were incubated at 37° C. in humidified atmosphere of 5% carbondioxide and 95% of air for 68 hours and pulsed with ³ H-thymidine (0.5μCi) 4 hours before the cells were collected. The object compound ofthis invention was dissolved in ethanol and further diluted in RPMI 1640medium and added to the cultures to give final concentrations of 0.1μg/ml or less.

The results are shown in Tables 7 to 10. The tricyclo compounds of thepresent invention suppressed mouse MLR.

                  TABLE 7                                                         ______________________________________                                        Effect of the FR-900506 Substance on MLR                                        FR-900506                                                                     concentration Radioactivities Suppression IC                                                                      .sub.50                                   (ng/ml) (mean C.P.M. ± S.E.) (%) (ng/ml)                                 ______________________________________                                        2.5       54 ± 4      99.5                                                   1.25 168 ± 23 98.3                                                         0.625 614 ± 57 93.8                                                        0.313 3880 ± 222 60.9 0.26                                                 0.156 5490 ± 431 44.7                                                      0.078 7189 ± 365 27.6                                                      0 9935 ± 428 0                                                           ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of the FR-900520 Substance on MLR                                        FR-900520                                                                     concentration Radioactivities Suppression IC                                                                      .sub.50                                   (ng/ml) (mean C.P.M. ± S.E.) (%) (ng/ml)                                 ______________________________________                                        100       175 ± 16    99.2                                                   10 515 ± 55 97.8                                                           1 2744 ± 527 88.1 0.38                                                     0.500  9434 ± 1546 59.2                                                    0.25 14987 ± 1786 35.1                                                     0 23106 ± 1652 0                                                         ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Effect of the FR-900523 Substance on MLR                                        FR-900523                                                                     concentration Radioactivities Suppression IC                                                                      .sub.50                                   (ng/ml) (mean C.P.M. ± S.E.) (%) (ng/ml)                                 ______________________________________                                        100        25 ± 12    99.9                                                   10 156 ± 37 99.3                                                           1 5600 ± 399 75.8 0.5                                                      0.500 11624 ± 395  49.7                                                    0.250 17721 ± 1083 23.3                                                    0 23106 ± 1052 0                                                         ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Effect of the FR-900525 Substance on MLR                                        FR-900525                                                                     concentration Radioactivities Suppression IC                                                                      .sub.50                                   (ng/ml) (mean C.P.M. ± S.E.) (%) (ng/ml)                                 ______________________________________                                        100       469 ± 56    97.0                                                   10 372 ± 32 97.6                                                           5  828 ± 369 94.7 1.55                                                     2.5 3564 ± 512 77.4                                                        1.2 10103 ± 421  35.8                                                      0 15741 ± 411  0                                                         ______________________________________                                    

Test 2

Antimicrobial activities of Tricyclo Compounds (I)

Antimicrobial activities of the tricyclo compounds (I) against variousfungi were determined by a serial agar dilution method in a Sabouraudagar. Minimum inhibitory concentrations (MIC) were expressed in terms ofμg/ml after incubation at 30° C. for 24 hours.

Tricyclo compounds of the present invention showed antimicrobialactivities against fungi, for example, Aspergillus fumigatus IFO 5840and Fusarium oxysporum IFO 5942 as described in the following Tables 11and 12.

                  TABLE 11                                                        ______________________________________                                        MIC values (μg/ml) of Tricyclo Compounds (I)                                 against Aspergillus fumigatus IFO 5840                                              Substances                                                                              MIC (μg/ml)                                              ______________________________________                                        FR-900506     0.025                                                             FR-900520 0.1                                                                 FR-900523 0.3                                                                 FR-900525 0.5                                                               ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        MIC values (μg/ml) of Tricyclo Compounds (I)                                 of against Fusarium oxysporum                                                       Substances                                                                              MIC (μg/ml)                                              ______________________________________                                        FR-900506     0.05                                                              FR-900525 1                                                                 ______________________________________                                    

Test 3

Effect of Tricyclo Compounds (1) on Skin Allograft Survival in Rats

Vental allografts from donor (Fischer) rats were grafted onto thelateral thoracic area of recipient (WKA) rats. The dressings wereremoved on day 5. The grafts were inspected daily until rejection whichwas defined as more than 90% necrosis of the graft epitherium.

The FR-900506 substance was dissolved in olive oil and administeredintramuscularly for 14 consecutive days, beginning at the day oftransplantation.

As shown in Table 13, all skin allografts were rejected within 8 days inrats treated with olive oil intramuscularly for 14 consecutive days, butdaily treatment with the FR-900506 substance clearly prolonged skinallograft survival.

                  TABLE 13                                                        ______________________________________                                        Effect of FP-9D0506 Substance on Skin Allograft Survival                                          Number of Skin Allograft                                    Dose (mg/kg) Animals Survival Day                                           ______________________________________                                        Control --          11        7,7,7,7,7,7,8,8,8,8,8                             (olive oil)                                                                   FR-900506 1 8 19,19,19,20,21,21,22,22                                         Substance 3.2 6 22,23,23,26,27,35                                              10 5 56,61,82,85,89                                                        ______________________________________                                    

Test 4

Effect of Tricylo Compounds (I) on Type II Collagen-Induced-Arthritis inRats

Collagen was dissolved in cold 0.01 M acetic acid at a concentration of2 mg/ml. The solution was emulsified in an equal volume of incompleteFreund's adjuvant. A total volume of 0.5 ml of the cold emulsion wasinjected intradermally at several sites on the back and one or two sitesinto the tail of female Lewis rats. The FR-900506 substance wasdissolved in olive oil and administered orally. Control rats immunizedwith same amount of type II collagen received oral administrations ofolive oil alone. Incidences of the arthritis were observed.

The test results are shown in Table 14. The inflammatory polyarthritiswas induced in all rats treated with olive oil for 14 days starting onthe same day as the type II collagen immunization.

Daily treatment with the FR-900506 substance for 14 days gave completesuppression of arthritis induction during an observation period of 3weeks.

                  TABLE 14                                                        ______________________________________                                        Effect of FR-900506 Substance on Type II                                        Collagen-induced-Arthritis in Rats                                                                     Incidence of                                         Dose (mg/kg per day) Arthritis                                              ______________________________________                                        Control      --            5/5                                                  (olive oil)                                                                   FR-900506 3.2 0/5                                                             Substance                                                                   ______________________________________                                    

Test 5

Effect of Tricylo Compounds (I) on Experimental AllergicEncephalomyelytis (EAE) in SJL/J Mice

Spinal cord homogenate was prepared from SJL/J mice. The spinal cordswere removed by insufflation, mixed with an approximately equal volumeof water and homogenized at 4° C. An equal volume of this coldhomogenate (10 mg/ml) was emulsified with complete Freund's adjuvant(CFA) containing 0.6 mg/ml of Mycobacterium tuberculosis H37RA.

EAE was induced by two injections of 0.2 ml of spinal cord-CFA emulsioninto SJL/J mice on day 0 and day 13. All mice used in these tests wereevaluated and scored daily for clinical signs of EAE.

The severity of EAE was scored according to the following criteria:grade 1-decreased tail tone: grade 2-a clumsy gait: grade 3-weakness ofone or more limb: grade 4-paraplegia or hemiplegia.

The FR-900506 substance was dissolved in olive oil and administeredorally for 19 days starting on day 0 (the day of first immunization). Asshown in Table 15, the FR-900506 substance clearly prevented thedevelopment of clinical signs of EAE.

                  TABLE 15                                                        ______________________________________                                        Effect of FR-900506 Substance on Experimental                                   Allergic Encephalomyelytis in SJL/J Mice                                                           Number of Animals                                        Dose (mg/kg) with Disease at Day 24                                         ______________________________________                                        Control     --         10/10                                                    (olive oii)                                                                   FR-900506 32 0/5                                                              Substance                                                                   ______________________________________                                    

Test 6

Effect of Tricyclo Compounds (I) on Local Graft-versus-Host Reaction(GvHR) in Mice

The viable spleen cells (1×10⁷ cells) from C57BL/6 donors were injectedsubcutaneously into the right hind foot pad of BDF₁ mice to induce localGvHR. The mice were killed 7 days later and both right (injected paw)and left (uninjected paw) popliteal lymph nodes (PLN) were weighed. TheGvHR was expressed as the weight difference between right and left PLN.

The FR-900506 substance was dissolved in olive oil and administeredorally for five days starting on the same day as sensitization.

ED₅₀ Value of the FR-900506 substance for prevention of the localgraft-versus-host reaction was 19 mg/kg.

Test 7

Acute toxicities of Tricyclo Compounds (I)

Test on acute toxicities of the FR-900506, FR-900520, FR-900523 andFR-900525 substances in ddY mice by intraperitoneal injection wereconducted, and the dead at dose of 100 mg/kg could not be observed ineach case.

Test 8

Effect of Tricyclo Compounds (I) on Antibody Formation in mice (Assayfor the haemagglutination test)

Male BDF₁ mice 6 to 8 weeks of age were used for this test. The micewere immunized on day 0 with 1×10⁸ sheep erythrocytes (SRBC)intraperitoneally. Serum aggulutinin titers were determined for eachindividual animal with sedimentation of erythrocytes by serial two folddilutions. The titers are expressed as -log₂.

The FR-900506 substance was administered orally for 5 days from one daybefore immunization to day 3.

The results of each experiments were evaluated by means of Student'st-test as shown in Table 16.

                  TABLE 16                                                        ______________________________________                                        Effect of FR-900506 Substance on antibody formation in mice                           Dose     Number    Haemagglutination                                                                        ED.sub.50                                 (mg/kg) of Animals Mean log.sub.2 titer ± SE (mg/kg)                     ______________________________________                                        Control --       5         8.2 ± 0.2                                                                             --                                        FR-900506 10 5 7.6 ± 0.4  16.9                                              32 5 6.0 ± 0.45                                                            100 5 4.4 ± 0.75                                                        ______________________________________                                    

Test 9

Effect of Tricyclo Compounds (I) on in vivo Plaque Forming Cell (PFC)Response in Mice

Male C3H/He mice 6 to 7 weeks of age were used for this test. The micewere immunized on day 0 with 0.2 ml of 1×10⁸ washed sheep erythrocytesintravenously. Spleens were removed on day 4 and spleen cells wereincubated in the presence of SRBC as described by Cunningham andSzenberg (1968). Tests were evaluated by enumeration of direct generatedplaque forming cells in the presence of complement. Suspensions ofspleen cells were counted with a Microcellcounter CC-130 (Sysmex, Japan)and PFC results were calculated as PFC/10⁶ recovered cells.

The FR-900506 substance was administered orally for 4 days starting fromthe immunization.

The results of each experiment were evaluated by means of Student'st-test as shown in Table 17.

                  TABLE 17                                                        ______________________________________                                        Effect of FR-900506 Substance on in vivo PFC Response in Mice                          Dose     Number     PFC/10.sup.6                                                                           ED.sub.50                                 (mg/kg) of Animals cells (mg/kg)                                            ______________________________________                                        Control  --       5          5054 ± 408                                                                          --                                        FP-900506 3.2 5 3618 ± 476 5.9                                              10 5 1382 ± 243                                                            32 5 657 ± 41                                                              100 5 278 ± 41                                                          ______________________________________                                    

Test 10

Effect of Tricyclo Compounds (I) on Contact-Delayed Hypersensitivity inmice

Female ICR mice were used for this test. A tenth of a 7% (w/v) solutionof picryl chloride in ethanol prepared at the time of the experiment wasapplied to the ventral surface of previously shaved animals. Such asensitization was performed twice at intervals of 7 days. Seven dayslater, the thickness of the ears were measured with an ordinaryengineer's micrometer and a 1% (w/v) solution of picryl chloride inolive oil was painted to both surfaces of each ear. The inflammation wasevaluated 24 hours later by measuring the both of ears. The results wereexpressed as the average increase in thickness of the ears measured inunits of 10⁻³ cm.

The FR-900506 substance was injected for 14 days orally, starting fromthe first sensitization.

Statistical significance was evaluated by Student's t-test as shown inTable 18.

                  TABLE 18                                                        ______________________________________                                        Effect of FR-900506 Substance on contact-delayed                                hypersensitivity in mice                                                             Dose     Number     Increase of ears at 24 hours                       (mg/kg) of Animals after challenge (×10.sup.-3 cm)                    ______________________________________                                        Control  --       5          15.5 ± 0.8                                      FR-900506 32 5 6.4 ± 1.6                                                    100 5 2.2 ± 1.5                                                         ______________________________________                                    

Test 11

Effect of Tricyclo Compounds (I) on Delayed Type Hypersensitivity (DTH)Response to Methylated Bovine Serum Albumin (MBSA)

Female BDF1 mice were used for this test. The mice were sensitized witha subcutaneous injection of 0.1 ml emulsion consisting of equal volumeof MBSA (2 mg/ml) and Freund's incomplete adjuvant (FIA). Seven dayslater, a 0.05 ml challenge dose of 0.4 mg/ml MBSA in saline was injectedinto the plantar region of the right hind foot and 0.05 ml saline intothe left hind foot to act as a control. Twenty four hours afterchallenge, both hind feet were measured with a dial gauge and the meanchallenge in footpad thickness was measured.

The FR-900506 substance was injected for 8 days orally, starting fromthe sensitization.

Statistical significance was evaluated by Student's t-test as shown inTable 19.

                  TABLE 19                                                        ______________________________________                                        Effect of FR-900506 Substance on DTH to MBSA in mice                                                        Footpad                                           Dose Number Swelling ED.sub.50                                                (mg/kg) of Animals (×10.sup.-3 cm) (mg/kg)                            ______________________________________                                        Control   --      5           57.4 ± 3.4                                                                        --                                         FR-900506 10 5 31.0 ± 4.4 18.4                                              32 5 20.8 ± 4.4                                                            100 5  7.8 ± 1.6                                                        ______________________________________                                    

Test 12

Inhibition of Interleukin-2 (IL-2) Production by Tricyclo Compounds (I)

The mixed lymphocyte reaction (MLR) was performed in microtiter plates,with each well containing 5×10⁵ C57BL/6 responder cells (H-2^(b)), 5×10⁵mitomycin C treated (25 μg/ml mitomycin C at 37° C. for 30 minutes andwashed three times with RPMI 1640 medium) BALB/C stimulator cells(H-2^(b)) in 0.2 ml RPMI 1640 medium supplemented with 10% fetal calfserum, 2 mM sodium hydrogen carbonate, penicillin 20 (50 unites/ml) andstreptomycin (50 μg/ml). The cells were incubated at 37° C. inhumidified atmosphere of 5% carbon dioxide and 95% of air for 48 hoursand the supernatant was collected. The FR-900506 substance was dissolvedin ethanol and further diluted in RPMI 1640 medium and added to thecultures to give final concentrations of 0.1 μg/ml or less.

The IL-2 activity was measured according to the method described by S.Gillis et. al. [Journal of Immunology, Vol. 120, page 2027, (1978)]. TheIL-2 dependent CTLL-2 cell line was used to quantitate IL-2 activity.Cells (5×10³ /well) were cultured at 37° C. for 24 hours with variousdilutions of IL-2 containing supernatant from MLR. The uptake of ³H-thymidine was measured by pulsing cultures with 0.5 μCi of ³H-thymidine for 6 hours. The unit value was calculated by dilutionanalysis of test sample and was compared with a laboratory standardpreparation in which 100 units are equivalent to the amount of IL-2necessary to achieve 50% proliferation of CTLL-2 cells.

As shown in Table 20, the FR-900506 substance of the present inventioninhibited the production of IL-2 from mouse MLR.

                  TABLE 20                                                        ______________________________________                                        Effect of the FR-900506 Substance on IL-2 Production                            FR-900506                                                                     concentration IL-2                                                            (nM) (units/ml) Inhibition %                                                ______________________________________                                        10              0        100                                                    1 0 100                                                                       0.3 0.2 99.5                                                                  0.1 25.0 34.4                                                                 0 38.1 --                                                                   ______________________________________                                    

The FR-900506 substance also inhibited lymphokine production such asinterleukin 3 and interferones, especially γ-interferone, in thesupernatant of MLR.

The pharmaceutical composition of this invention can be used in the formof a pharmaceutical preparation, for example, in solid, semisolid orliquid form, which contains the tricyclo compounds (I) of the presentinvention, as an active ingredient, in admixture with an organic orinorganic carrier or excipient suitable for external, enteral orparenteral applications. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The carriers which canbe used are water, glucose, lactose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form,and in addition auxiliary, stabilizing, thickening and coloring agentsand perfumes may be used. The active object compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of diseases.

For applying this composition to human, it is preferable to apply it byparenteral or enteral administration. While the dosage oftherapeutically effective amount of the tricyclo compounds (I) variesfrom and also depends upon the age and condition of each individualpatient to be treated, a daily dose of about 0.01-1000 mg, preferably0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient isgenerally given for treating diseases, and an average single dose ofabout 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg isgenerally administered.

The following examples are given for the purpose of illustrating thepresent invention.

EXAMPLE 1

Isolation of Streptomyces tsukubaensis No. 9993

Streptomyces tsukubaensis No. 9993 was isolated by using dilution platetechniques as shown in the following.

About one gram soil which was collected at Toyosato-cho, Tsukuba Gun,Ibaraki Prefecture, Japan, was added to a sterile test tube and thevolume made up to 5 ml with sterile water. The mixture was then blendedfor 10 second by a tube buzzer and kept on 10 minutes. The supernatantwas sequentially diluted by 100 fold with sterile water. The dilutedsolution (0.1 ml) was spread on Czapek agar supplemented with thiaminehydrochloride (saccharose 30 g, sodium nitrate 3 g, dipotassiumphosphate 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g,ferrous sulfate 0.01 g, thiamine hydrochloride 0.1 g, agar 20 g, tapwater 1000 ml; pH 7.2) in a Petri dish. The growing colonies developedon the plates after 21 days incubation at 30° C. were transferred toslants [yeast-malt extract agar (ISP-medium 2)], and cultured for 10days at 30° C. Among of the colonies isolated, the Streptomycestsukubaensis No. 9992 could be found.

Fermentation

A culture medium (160 ml) containing glycerin (1%), soluble starch (1%),glucose (0.5%), cottonseed meal (0.5%), dried yeast (0.5%), corn steepliquor (0.5%) and Calcium carbonate (0.2%) (adjusted to pH 6.5) waspoured into each of twenty 500 ml-Erlenmeyer flasks and sterilized at120° C. for 30 minutes. A loopful of slant culture of Streptomycestsukubaensis No.9993, FERM BP-927 was inoculated to each of the mediaand cultured at 30° C. for 4 days on a rotary shaker. The resultantculture was inoculated to a medium containing soluble starch (4.5%),corn steep liquor (1%), dried yeast (1%), calcium carbonate (0.1%) andAdekanol (defoaming agent, Trade Mark, maker; Asahi Denka Co.) (0.1%)(150 liters) in a 200-liter jar-fermentor, which had been sterilized at120° C. for 20 minutes in advance, and cultured at 30° C. for 4 daysunder aeration of 150 liters/minutes and agitation of 250 rpm.

Isolation and Purification

The cultured broth thus obtained was filtered with an aid ofdiatomaseous earth (5 kg). The mycelial cake was extracted with methanol(50 liters), yielding 50 liters of the extract. The methanol extractfrom mycelium and the filtrate were combined and passed through a columnof a non-ionic adsorption resin "Diaion HP-20" (Trade Mark, makerMitsubishi Chemical Industries Ltd.) (10 liters). After washing withwater (30 liters) and aqueous methanol (30 liters), elution was carriedout with methanol. The eluate was evaporated under reduced pressure togive residual water (2 liters). This residue was extracted with ethylacetate (2 liters). The ethyl acetate extract was concentrated underreduced pressure to give an oily residue. The oily residue was mixedwith twice weight of acidic silica gel (special silica gel grade 12,maker Fuji Devison Co.), and this mixture was slurried in ethyl acetate.After evaporating the solvent, the resultant dry powder was subjected tocolumn chromatography of the same acid silica gel (800 ml) which waspacked with n-hexane. The column was developed with n-hexane (3 liters),a mixture of n-hexane and ethyl acetate (9:1 v/v, 3 liters and 4:1 v/v,3 liters) and ethyl acetate (3 liters). The fractions containing theobject compound were collected and concentrated under reduced pressureto give an oily residue. The oily residue was dissolved in a mixture ofn-hexane and ethyl acetate (1:1 v/v, 30 ml) and subjected to columnchromatography of silica gel (maker Merck Co., Ltd. 230-400 mesh) (500ml) packed with the same solvents system.

Elution was carried out with a mixture of n-hexane and ethyl acetate(1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters). Fractions containing thefirst object compound were collected and concentrated under reducedpressure to give a yellowish oil. The oily residue was mixed twiceweight of acidic silica gel and this mixture was slurried in ethylacetate. After evaporating the solvent, the resultant dry powder waschromatographed on acidic silica gel packed and developed with n-hexane.Fractions containing the object compound were collected and concentratedunder reduced pressure to give crude FR-900506 substance (1054 mg) inthe form of white powder.

100 mg of this crude product was subjected to high performance liquidchromatography. Elution was carried out using a column (8φ×500 mm) withLichrosorb SI 60 (Trade Mark, made by Merck & Co.) as a carrier. Thischromatography was monitored by UV detector at 230 nm and mobile phasewas a mixture of methylene chloride and dioxane (85:15 v/v) under flowrate of 5 ml/minute., and the active fractions were collected andevaporated. This high performance chromatography was repeated again, and14 mg of the purified FR-900506 substance was obtained as white powder.

Further, elution was continuously carried out with ethyl acetate (1.5liters), and fractions containing the second object compound werecollected and concentrated under reduced pressure to give crudeFR-900525 substance (30 mg) in the form of yellowish oil.

EXAMPLE 2

Fermentation

A preculture medium (100 ml) containing glycerin (1%), corn starch (1%),glucose (0.5%), cottenseed meal (1%), corn steep liquor (0.5%), driedyeast (0.5%) and calcium carbonate (0.2%) at pH 6.5 was poured into a500 ml-Erlenmeyer flask and sterilized at 120° C. for 30 minutes. Aloopful of slant culture of Streptomyces tsukubaensis No. 9993 wasinoculated to the medium and cultured at 30° C. for four days. Theresultant culture was transferred to the same preculture medium (20liters) in 30 liters jar-fermentor which had been sterilized at 120° C.for 30 minutes in advance. After the culture was incubated at 30° C. for2 days, 16 liters of the preculture was inoculated to a fermentationmedium (1600 liters) containing soluble starch (4.5%), corn steep liquor(1%), dried yeast (1%), calcium carbonate (0.1%) and Adekanol (defoamingagent, Trade Mark, maker Asahi Denka Co.) (0.1%) at pH 6.8 in 2 ton tankwhich had been sterilized at 120° C. for 30 minutes in advance andcultured at 30° C. for 4 days.

Isolation and Purification

The cultured broth thus obtained was filtered with an aid ofdiatomaseous earth (25 kg). The mycelial cake was extracted with acetone(500 liters), yielding 500 liters of the extract. The acetone extractfrom mycelium and the filtrate (1350 liters) were combined and passedthrough a column of a non-ionic adsorption resin "Diaion HP-20" (TradeMark, maker Mitsubishi Chemical Industries Ltd.) (100 liters). Afterwashing with water (300 liters) and 50% aqueous acetone (300 liters),elution was carried out with 75% aqueous acetone. The eluate wasevaporated under reduced pressure to give residual water (300 liters).This residue was extracted with ethyl acetate (20 liters) three times.The ethyl acetate extract was concentrated under reduced pressure togive an oily residue. The oily residue was mixed with twice weight ofacidic silica gel (special silica gel grade 12, maker Fuji Devison Co.),and this mixture was slurried in ethyl acetate. After evaporating thesolvent, the resultant dry powder was subjected to column chromatographyof the same acidic silica gel (8 liters) which was packed with n-hexane.The column was developed with n-hexane (30 liters), a mixture ofn-hexane and ethyl acetate (4:1 v/v, 30 liters) and ethyl acetate (30liters). The fractions containing the object compound were collected andconcentrated under reduced pressure to give an oily residue. The oilyresidue was mixed with twice weight of acidic silica gel and thismixture was slurried in ethyl acetate. After evaporating the solvent,the resultant dry powder was rechromatographed on acidic silica gel (3.5liters) packed with n-hexane. The column was developed with n-hexane (10liters), a mixture of n-hexane and ethyl acetate (4:1 v/v, 10 liters)and ethyl acetate (10 liters). Fractions containing the object compoundwere collected and concentrated under reduced pressure to give ayellowish oil. The oily residue was dissolved in a mixture of n-hexaneand ethyl acetate (1:1 v/v, 300 ml) and subjected to columnchromatography of silica gel (maker Merck Co., Ltd. 230-400 mesh) (2liters) packed with the same solvents system. Elution was curried outwith a mixture of n-hexane and ethyl acetate (1:1 v/v, 10 liters and 1:2v/v 6 liters) and ethyl acetate (6 liters).

Fractions containing the first object compound were collected andconcentrated under reduced pressure to give FR-900506 substance in theform of white powder (34 g). This white powder was dissolved inacetonitrile and concentrated under reduced pressure. This concentratewas kept at 5° C. overnight and prisms (22.7 g) were obtained.Recrystallization from the same solvent gave purified FR-900506substance (13.6 g) as colorless prisms.

Further, fractions containing the second object compound were collectedand concentrated under reduced pressure to give crude FR-900525substance (314 mg) in the form of yellowish powder.

EXAMPLE 3

Fermentation

A culture medium (160 ml) containing glycerin (1%), corn starch (1%),glucose (0.5%), cottonseed meal (1%), dried yeast (0.5%), corn steepliquor (0.5%) and calcium carbonate (0.2%) (adjusted to pH 6.5) waspoured into each of ten 500 ml-Erlenmeyer flasks and sterilized at 120°C. for 30 minutes. A loopful of slant culture of Streptomycestsukubaensis No. 9993 was inoculated to each of the medium and culturedat 30° C. for 4 days on a rotary shaker. The resultant culture wasinoculated to a medium containing soluble starch (5%), peanut powder(0.5%), dried yeast (0.5%), gluten meal (0.5%), calcium carbonate (0.1%)and Adekanol (deforming agent, Trade Mark, maker Asasi Denka Co.) (0.1%)(150 liters) in a 200-liter jar-fermentor, which had been sterilized at120° C. for 20 minutes in advance, and cultured at 30° C. for 4 daysunder aeration of 150 liters/minutes and agitation of 250 rpm.

Isolation and Purification

The cultured broth thus obtained was filtered with an aid ofdiatomaseous earth (5 kg). The mycelial cake was extracted with acetone(50 liters), yielding 50 liters of the extract. The acetone extract frommycelium and the filtrate (135 liters) were combined and passed througha column of a non-ionic adsorption resin "Diaion HP-20" (Trade Mark,maker Mitsubishi Chemical Industries Ltd.) (10 liters). After washingwith water (30 liters) and 50% % aqueous acetone (30 liters), elutionwas carried out with 75% aqueous acetone. The eluate (30 liters) wasevaporated under reduced pressure to give residual water (2 liters).This residue was extracted with ethyl acetate (2 liters) three times.The ethyl acetate extract was concentrated under reduced pressure togive an oily residue. The oily residue was mixed with twice weight ofacidic silica gel (special silica gel grade 12, maker Fuji Devison Co.),and this mixture was slurried in ethyl acetate. After evaporating thesolvent, the resultant dry powder was subjected to column chromatographyof the same acidic silica gel (800 ml) which was packed with n-hexane.The column was developed with n-hexane (3 liters), a mixture of n-hexaneand ethyl acetate (4:1 v/v, 3 liters) and ethyl acetate (3 liters). Thefractions containing the object compound were collected and concentratedunder reduced pressure to give an oily residue. The oily residue wasdissolved in a mixture of n-hexane and ethyl acetate (1:1 v/v, 30 ml)and subjected to column chromatography of silica gel (maker Merck Co.,Ltd. 230-400 mesh) (500 ml) packed with the same solvents system.Elution was carried out with a mixture of n-hexane and ethyl acetate(1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters) and ethyl acetate (1.5liters).

Fractions containing the first object compound were collected andconcentrated under reduced pressure to give crude FR-900506 substance (3g) in the form of yellowish powder.

Further, fractions containing the second object compound were collectedand concentrated under reduced pressure to give an oily residue. Thisoily residue was rechromatographed with silica gel to give a yellowishoil. The oily residue was mixed with twice weight of acidic silica geland this mixture was slurried in ethyl acetate. After evaporating thesolvent, the resultant dry powder was chromatographed on acidic silicagel (100 ml) packed and developed with n-hexane. Fractions containingthe object compound were collected and concentrated under reducedpressure to give FR-900525 substance in the form of pale yellowishpowder (380 mg). This powder was dissolved in a 1 mixture of n-hexaneand ethyl acetate (1:2 v/v, 5 ml) and subjected to acidic silica gel(special silica gel grade 922, maker Fuji Devison Co.) (100 ml) packedand washed with the same solvent system. Elution was carried out withethyl acetate. The active fractions were collected and evaporated underreduced pressure to give the purified FR-900525 substance (230 mg) inthe form of white powder.

EXAMPLE 4

Isolation of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238

Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 was isolatedby using dilution plate techniques as shown in the following.

About one gram soil which was collected at Yakushima, KagoshimaPrefecture, Japan, was added to a sterile test tube and the volume madeup to 5 ml with sterile water. The mixture was then blended for 10seconds by a tube buzzer and kept on 10 minutes. The supernatant wassequentially diluted by 100 fold with sterile water. The dilutedsolution (0.1 ml) was spread on Czapek agar supplemented with thiaminehydrochloride (saccharose 30 g, sodium nitrate 3 g, dipotassiumphosphate 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g,ferrous sulfate 0.01 g, thiamine hydrochloride 0.1 g, agar 20 g, tapwater 1000 ml; pH 7.2) in a Petri dish. The growing colonies developedon the plates after 21 days incubation at 30° C. were transferred toslants [yeast-malt extract agar (ISP-medium 2)], and cultured for 10days at 30° C. Among of the colonies isolated, the Streptomyceshygroscopicus subsp. yakushimaensis No. 7238 could be found.

Fermentation

A culture medium (160 ml) containing glycerin (1%), soluble starch (1%),glucose (0.5%), cottonseed meal (0.5%), dried yeast (0.5%), corn steepliquor (0.5%) and calcium carbonate (0.2%) (adjusted to pH 6.5) waspoured into each of twenty 500 ml-Erlenmeyer flasks and sterilized at120° C. for 30 minutes. A loopful of slant culture of Streptomyceshygroscopicus subsp. yakushimaensis No. 7238, FERM BP-928 was inoculatedto each of the media and cultured at 30° C. for 4 days on a rotaryshaker. The resultant culture was inoculated to a medium containingglucose (4.5%), corn steep liquor (1%), dried yeast (1%), gluten meal(1%), wheat germ (0.5%), calcium carbonate (0.1%) and Adekanol(defoaming agent, Trade Mark, maker Asahi Denka Co.) (0.1%) (150 liters)in a 200-liter jar-fermentor, which had been sterilized at 120° C. for20 minutes in advance, and cultured at 30° C. for 4 days under aerationof 150 liters/minutes and agitation of 250 rpm.

Isolation and Purification

The cultured broth thus obtained was filtered with an aid ofdiatomaseous earth (5 kg). The mycelial cake was extracted with acetone(50 liters), yielding 50 liters of the extract. The acetone extract frommycelium and the filtrate (135 liters) were combined and passed througha column of a non-ionic adsorption resin "Diaion HP-20" (Trade Mark,maker Mitsubishi Chemical Industries Ltd.) (10 liters). After washingwith water (30 liters) and aqueous acetone (30 liters), elution wascarried out with acetone. The eluate was evaporated under reducedpressure to give residual water (2 liters). This residue was extractedwith ethyl acetate (4 liters). The ethyl acetate extract wasconcentrated under reduced pressure to give an oily residue. The oilyresidue was mixed with twice weight of acidic silica gel (special silicagel grade 12, maker Fuji Devison Co.), and this mixture was slurried inethyl acetate. After evaporating the solvent, the resultant dry powderwas subjected to column chromatography of the same acid silica gel (800ml) which was packed with n-hexane. The column was developed withn-hexane (3 liters), a mixture of n-hexane and ethyl acetate (4:1 v/v, 3liters) and ethyl acetate (3 liters). The fractions containing theFR-900520 and FR-900523 substances were collected and concentrated underreduced pressure to give an oily residue. The oily residue was dissolvedin a mixture of n-hexane and ethyl acetate (1:1 v/v 50 ml) and subjectedto column chromatography of silica gel (maker Merck Co., Ltd. 70-230mesh) (1000 ml) packed with the same solvents system. Elution wascarried out with a mixture of n-hexane and ethyl acetate (1:1 v/v, 3liters and 1:2 v/v, 3 liters) and ethyl acetate (3 liters). Fractionscontaining the object compounds were collected and concentrated underreduced pressure to give a yellowish powder (4.5 g). This powder wasdissolved in methanol (20 ml) and mixed with water (10 ml). The mixturewas chromatographed on a reverse chase silica gel "YMC" (60-200 mesh)(500 ml) (Trade Mark, maker Yamamura Chemical institute) packed anddeveloped with a mixture of methanol and water (4:1 v/v).

Fractions containing the FR-900520 substance were collected andconcentrated under reduced pressure to give crude product of theFR-900520 substance (1.8 g) in the form of pale yellowish powder. Thispowder was dissolved in a small amount of diethyl ether. After standingovernight, the precipitated crystals were collected by filtration,washed with diethyl ether and then dried under reduced pressure.Recrystallization from diethyl ether gave 600 mg of the purifiedFR-900520 substance in the form of colorless plates.

The chromatography of the reverse phase silica gel was carried on withthe same solvents system, and the subsequent fractions containing theFR-900523 substance were collected and then concentrated under reducedpressure to give crude product of the FR-900523 substance (0.51 g) inthe form of pale yellowish powder. This crude product was dissolved inacetonitrile (3 ml) and subjected to a reverse phase silica gel "YMC"(70 ml) packed and developed with a mixture of acetonitrile,tetrahydrofuran and 50 mM phosphate buffer solution (pH 2.0) (3:2:5,v/v). Fractions containing the object compound were collected and wereextracted with ethyl acetate. This extract was concentrated underreduced pressure to give a yellowish white powder (190 mg). Theyellowish white powder was chromatographed again on a reverse phasesilica gel "YMC" to give white powder (80 mg) This white powder wasdissolved in a small amount of diethyl ether and allowed to standovernight at room temperature to give 56 mg of crystals.Recrystallization from diethyl ether gave 34 mg of the FR-900523substance in the form of colorless needles.

EXAMPLE 5

To a solution of the FR-900506 substance (10.4 mg) in dichloromethane(0.2 ml) were added pyridine (0.1 ml) and acetic anhydride (0.05 ml) atroom temperature, and the mixture was stirred for 5 hours. The solventwas removed from the reaction mixture under reduced pressure. Theresidue was subjected to silica gel thin layer chromatography(developing solvent: diethyl ether and dichloromethane, 1:2 v/v) to give12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methylvinyl]-17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (6.0 mg).

IR ν(CHCl₃): 3520, 1728, 1705(sh), 1640, 1095 cm⁻¹.

EXAMPLE 6

To a solution of the FR-900506 substance (52.5 mg) in dichloromethane (1ml) were added pyridine (0.5 ml) and acetic anhydride (0.3 ml) at roomtemperature, and the mixture was stirred at room temperature for 9hours. The solvent was removed from the reaction mixture under reducedpressure. The residue was subjected to silica gel thin layerchromatography (developing solvent: diethyl ether and hexane, 3:1 v/v)to give14-acetoxy-12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methylvinyl]-17-allyl-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16tetraone (48.0 mg) and12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methylvinyl]-17-allyl-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.sup.4,9]octacosa-14,18-diene-2,3,10,16-tetraone (5.4 mg), respectively.

Former Compound

IR ν(CHCl₃): 1730, 1720(sh), 1640 cm⁻¹.

Latter Compound

IR ν(CHCl₃): 1730, 1690, 1640, 1627 cm⁻¹.

EXAMPLE 7

To a solution of the FR-900506 substance (9.7 mg) in dichloromethane(0.2 ml) and pyridine (0.1 ml) was added benzoyl chloride (50 μl) atroom temperature, and the mixture was stirred at room temperature for 2hours. The solvent was removed from the reaction mixture under reducedpressure to give a crude oil. This oil was purified on silica gel thinlayer chromatography (developing solvent: diethyl ether and hexane, 2.1v/v) to afford17-allyl-12-[2-(4-benzoyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (8.0 mg).

IR ν(CHCl₃): 3500, 1735(sh), 1710, 1640, 1600 cm⁻¹.

EXAMPLE 8

To a solution of the FR-900506 substance (30.5 mg) in pyridine (1 ml)was added p-nitrobenzoyl chloride (ca. 100 mg), and the mixture wasstirred at room temperature for 2 hours. The reaction mixture wasdiluted with ethyl acetate, and washed with a saturated aqueous sodiumhydrogen carbonate, water 1N-hydrochloric acid, water, a saturatedaqueous sodium hydrogen carbonate, water and an aqueous sodium chloride,successively, and then dried. The resulting solution was concentratedunder reduced pressure, and the residue was purified on silica gelcolumn chromatography to give17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-[4-(p-nitrobenzoyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (37.7 mg).

IR ν(CHCl₃): 1720, 1640, 1610, 1530-1520 cm⁻¹.

EXAMPLE 9

17-Allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-[4-(3,5-dinitrobenzoyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴.9]octacos-18-ene-2,3,10,16-tetraone (36.0 mg) was obtained by reactingthe FR-900506 substance (30.6 mg) with 3,5-dinitrobenzoyl chloride (33mg) in pyridine (0.5 ml) in accordance with a similar manner to that ofExample 8.

IR ν(CHCl₃): 1730, 1640, 1610, 1530-1520 cm⁻¹.

EXAMPLE 10

17-Allyl-1,14-dihydroxy-23,25-dimethoxy-12-[2-[4-(2-lmenthyloxyacetoxy)-3-methoxycyclohexyl]-1-methylvinyl]-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴.9]octacos-18-ene-2,3,10,16-tetraone (50.9 mg) was obtained by reactingthe FR-900506 substance (48 mg) with 2-l-menthyloxyacetyl chloride (0.08) in pyridine (0.05 ml) in accordance with a similar manner to that ofExample 8.

IR ν(neat): 3520, 1760, 1740(sh), 1720(sh), 1652 cm⁻¹.

EXAMPLE 11

To a solution of (-)-2-trifluoromethyl-2-methoxy-2-phenylacetic acid (51mg) in ethyl acetate (10 ml) was added at room temperatureN,N'-dicyclohexylcarbodiimide (47 mg). After stirring for 1.5 hours atroom temperature, then the FR-900506 substance (25.0 mg) and4-(N,N-dimethylamino)pyridine (11 mg) were added, followed by stirringat room temperature for 3.5 hours. The resulting solution wasconcentrated to provide a residue, which was taken up in diethyl etherand then washed successively with hydrochloric acid, an aqueous sodiumhydrogen carbonate and an aqueous sodium chloride. The organic layer wasdried over sodium sulfate and concentrated to provide a residue, whichwas chromatographed on silica gel (developing solvent: dichloromethaneand diethyl ether, 10:1 v/v) to give17-allyl-12-[2-[4-[(-)-2-trifluoromethyl-2-methoxy-2-phenylacetoxy]-3-methoxycyclohexyl]-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴.9]octacos-18-ene-2,3,10,16-tetraone (6.5 mg) and17-allyl-14-[(-)-2-trifluoromethyl-2-methoxy-2-phenylacetoxy]-12-[2-[4-[(-)-2-trifluoromethyl-2-methoxy-2-phenylacetoxy]-3-methoxycyclohexyl]-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (20.2 mg).

Former Compound

IR ν(neat): 3510, 1750, 1730(sh), 1710, 1652, 1500 cm⁻¹.

Latter Compound

IR ν(neat): 1750, 1720, 1652, 1500 cm⁻¹.

EXAMPLE 12

To a stirred solution of the FR-900506 substance (248 mg) in pyridine (7ml) were added succinic anhydride (145 mg) and4-(N,N-dimethylamino)pyridine (7 mg), and the resulting mixture wasstirred at room temperature for 18 hours. The reaction mixture wassubjected to chromatography on pressure and the residue was subjected tochromatography on silica gel (20 g) with ethyl acetate to give17-allyl-12-[2-[4-(3-carboxypropionyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16,-tetraone (90 mg).

IR ν(CHCl₃): 350, 3100-2300, 1720, 1705(sh), 1635 cm⁻¹.

EXAMPLE 13

To a solution of the FR-900506 substance (100.7 mg) in pyridine (3 ml)was added p-iodobenzenesulfonyl chloride (500 mg), and the mixture wasstirred at room temperature for 36 hours. The solution was diluted withethyl acetate and washed with a saturated aqueous sodium hydrogencarbonate, water and an aqueous sodium chloride. The organic layer wasdried over sodium sulfate, filtered and concentrated under reducedpressure. The residue was chromatographed on silica gel (developingsolvent: diethyl ether and hexane, 3:1 v/v) to give17-allyl-1,14-dihydroxy-12-[2-[4-(p-iodobenzenesulfonyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴.9]octacos-18-ene-2,3,10,16-tetraone (61 mg) and17-allyl-1-hydroxy-12-[2-[4-(p-iodobenzenesulfonyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28dioxa-4-azatricyclo[22.3.1.0⁴,9]octacosa-14,18-diene-2,3,10,16-tetraone (12 mg), respectively.

Former Compound

IR ν(CHCl₃): 3470, 1730, 1717, 1692, 1635, 1568 cm⁻¹.

Latter Compound

¹ H NMR δ ppm (CDCl₃): 6.15 (d, J=15 Hz) 6.25 (d, j=15Hz) 6.70 (dd,J=15Hz, 10Hz) 6.80 (dd, J=15Hz, 10Hz) 7.60 (2H, m), 7.90 (2H, m),

EXAMPLE 14

17Allyl-12-[2-[2-(4-d-camphorsulfonyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14,-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[2.3.1.0⁴,9]octacos-18ene-2,3,10,16-tetraone (34 mg) was obtained by reacting theFR-900506 substance (27 mg) with d-camphorsulfonyl chloride (97 mg) inpyridine (0.6 ml) in accordance with a similar manner to that of Example13.

IR ν(neat): 3500, 1747, 1720(sh), 1710(sh), 1655 cm⁻¹.

EXAMPLE 15

To a stirred solution of the FR-900506 substance (89.7 mg) indichloromethane (3 ml) were added imidazole (118 mg) andtert-butyl-diphenylsilyl chloride (52.2 mg). After the mixture wasstirred at room temperature for 2 hours, the reaction mixture wasdiluted with a saturated aqueous ammonium chloride and extracted threetimes with diethyl ether. The extract was washed with water and anaqueous sodium chloride, dried over sodium sulfate, and thenconcentrated under reduced pressure. The residue was purified on silicagel column chromatography (developing solvent: ethyl acetate and hexane,1:3 v/v) to give17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-2,3,10,16-tetraone (107 mg).

IR ν(neat): 3520, 1742, 1705, 1650 cm⁻¹.

EXAMPLE 16

17-Allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (85 mg) was obtained by reacting theFR-900506 substance (80 mg) with tert-butyl-dimethylsilyl chloride (17mg) in the presence of imidazole (15 mg) in N,N-dimethylformamide (1 ml)in accordance with a similar manner to that of Example 15.

IR ν(CHCl₃): 1735, 1720(sh), 1700, 1640 cm⁻¹.

EXAMPLE 17

To a solution of the FR-900506 substance (100 mg) in dimethyl sulfoxide(1.5 ml) was added acetic anhydride (1.5 ml) and the mixture was stirredat room temperature for 14 hours. The reaction mixture was diluted withethyl acetate and washed with a saturated aqueous sodium hydrogencarbonate, water and an aqueous sodium chloride. The organic layer wasdried over sodium sulfate, filtered and then concentrated under reducedpressure. The residue was subjected to thin layer chromatography onsilica gel (developing solvent: diethyl ether) to give17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4-methylthiomethoxy-3-methoxycyclohexyl)-1-methylvinyl]-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacosa-14,18-diene-2,3,10,16-tetraone (51 mg),17-allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacosa-14,18-diene-2,3,10,16-tetraone (18 mg) and17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4-methylthiomethoxy-3-methoxycyclohexyl)-1-methylvinyl]-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (10 mg), respectively.

First Compound

IR ν(CHCl₃): 3470, 1730, 1635, 1630(sh), 1580(sh) cm⁻¹.

Second Compound

IR ν(CHCl₃): 1728, 1640, 1090 cm⁻¹.

Third Compound

IR ν(CHCl₃): 3480, 1735, 1710, 1640 cm⁻¹.

EXAMPLE 18

To a solution of17-allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (39.9 mg) in pyridine (1.5 ml) wasadded acetic anhydride (0.5 ml), and the mixture was stirred at roomtemperature for 6 hours. The solvent was removed from the reactionmixture under reduced pressure to give a crude oil, which was purifiedon silica gel thin layer chromatography (developing solvent: diethylether and hexane, 1:1 v/v) to afford14-acetoxy-17-allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (26.5 mg).

IR ν(CHCl₃): 1728, 1715(sh), 1635 cm⁻¹.

EXAMPLE 19

14-Acetoxy-17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (10 mg) was obtained by reacting17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (10.6 mg) with acetic anhydride (0.1mg) in pyridine (0.2 ml) in accordance with a similar manner to that ofExample 18.

IR ν(CHCl₃): 3500, 1730, 1720(sh), 1660(sh), 1640, 1620(sh), 1100 cm⁻¹.

EXAMPLE 20

To a solution of14-acetoxy-17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (43.8 mg) in tetrahydrofuran (1.5 ml)was added potassium carbonate (ca 100 mg) at room temperature and themixture was stirred at the same temperature for 3 hours. The reactionmixture was diluted with diethyl ether and the resulting solution waswashed with a saturated aqueous ammonium chloride, water and an aqueoussodium chloride successively, and dried over sodium sulfate. Theresulting solution was concentrated under reduced pressure and theresidue was purified on silica gel thin layer chromatography (developingsolvent: diethyl ether and hexane, 3:2 v/v) to give17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,26-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacosa-14,18-diene-2,3,10,16-tetraone (30 mg).

IR ν(CHCl₃): 1733, 1720(sh), 1685, 1640(sh), 1620 cm⁻¹.

EXAMPLE 21

A solution of the FR-900506 substance (50 mg) in ethyl acetate (2 ml)was subjected -o catalytic reduction using 10% palladium on carbon (10mg) under atmospheric pressure at room temperature for 20 minutes. Thereaction mixture was filtered and the filtrate was evaporated todryness, which was purified on thin layer chromatography. Developmentwith a mixture of chloroform and acetone (5:1 v/v) gave1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-17-propyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (50.0 mg).

IR ν(CHCl₃): 3480, 1735(sh), 1717, 1700, 1650(sh), 1625 cm⁻¹.

EXAMPLE 22

White powder of crude FR-900506 substance (1 g) obtained by a similarfermentation process to Example 1 was dissolved in acetonitrile (5 ml)and subjected to high performance liquid chromatography (HPLC) usingShimazu LC4A (Trade Mark, made by Shimazu Seisaku-sho). Steel column (25mm inside diameter, 250 mm length) packed with YMC-S343 (ODS) (TradeMark, made by Shimakyu Co., Ltd.) was used at a flow rate of 12 ml/min.Mobile phase was an aqueous mixture of 28% acetonitrile 10% n-butanol,0.075% phosphoric acid, 3.75 mM sodium dodecyl sulfate (SDS) anddetection was carried out using Hitachi UV-recorder at 210 nm. Onehundred μl of the sample was injected each time and the HPLC wasrepeated 50 times so that all the sample could be subjected to thecolumn. Each eluate with a retention time of 85 min. to 90 min. wascollected and extracted with an equal volume of ethyl acetate (3.6liters). The ethyl acetate layer was separated and washed with anaqueous sodium hydrogen carbonate (1%, 2 liters) and concentrated invacuo to a small amount. SDS crystallized on concentration was removedby filtration. Crude powder obtained was dissolved in acetonitrile at aconcentration of 100 mg/ml and applied again to HPLC. Mobile phase wasan aqueous mixture of 12.5% acetonitrile, 9.75% n-butanol, 0.075%phosphoric acid, 3.75 mM SDS. The column was eluted at a flow rate of 10ml/min. The eluates with a retention time of 131 min. to 143 min. werecollected and extracted with equal volume of ethyl acetate. The solventlayer was separated and washed with 1% aqueous sodium hydrogen carbonateand concentrated in vacuo to a small volume. SDS crystallized onconcentration was removed by filtration.

Crude powder thus obtained was dissolved in a small amount of ethylacetate and subjected to column chromatography using silica gel (10 ml)(Kiesel gel, 230-400 mesh, maker: Merck Co., Ltd.). The column waswashed with a mixture of n-hexane and ethyl acetate (30 ml) (1:1 v/v)and a mixture of n-hexane and ethyl acetate (60 ml) (1:2 v/v). Elutionwas carried out using ethyl acetate and fractionated (each fraction: 3ml). Fractions 18 to 24 were collected and concentrated in vacuo todryness to give FR-900520 substance (24 mg).

EXAMPLE 23

A solution of the FR-900506 substance (310 mg), 2-trimethylsilylethyl4-isocyanatobutyrate (350 mg) and triethylamine (6 drops) in anhydrousbenzene (4 ml) was heated at 50° C. with stirring for 2 hours. Thereaction mixture was allowed to stand at room temperature overnight. Themixture was concentrated to dryness under reduced processure to leave aresidue, which was chromatographed on silica gel in chloroform. Elutionwas carried out with chloroform to give17-allyl-12-[2-[3-methoxy-4-{3-(2-trimethylsilylethoxycarbonyl)propylcarbamoyloxy}cyclohexyl]-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone (99 mg). This product was treatedwith tetra(n-butyl)ammonium fluoride (0.12 m mole) in tetrahydrofuran(2.5 ml) at room temperature for 20 minutes. The reaction mixture wasconcentrated to dryness under reduced pressure to leave a residue, whichwas purified with preparative thin layer chromatography on silica gel.Elution with a mixture of chloroform and methanol (5:1) gave17-allyl-12-[2-{4-(3-carboxypropylcarbamoyloxy)-3-methoxycyclohexyl}-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone (18.1 mg).

IR ν(CHCl₃) 3550-3100, 2870, 2750-2400, 1730, 1690, 1630 cm⁻¹.

What we claim is:
 1. A method for suppressing immune reaction,comprising:administering to a subject in need thereof an effectiveamount of a compound of formula (I): ##STR10## wherein R¹ is hydroxy orpharmaceutically acceptable protected hydroxy selected from the groupconsisting of 1-(lower alkylthio)(lower)alkyloxy,tri(lower)alkylsilyloxy, lower alkyldiphenyl-silyloxy, pharmaceuticallyacceptable organic carboxylic acyloxy, pharmaceutically acceptableorganic sulfonic acyloxy, and pharmaceutically acceptable organiccarbamic acyloxy, R² is hydrogen or the same meanings as R¹, R³ ismethyl, ethyl, propyl or allyl, n is an integer of 1 or 2, and thesymbol of a line and dotted line is a single bond or a double bond, orpharmaceutically acceptable basic salt thereof.
 2. The method accordingto claim 1, wherein said compound of formula (I)is:17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone.
 3. The method according to claim 1,wherein said compound of formula (I)is:17-ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone.